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G-Protein and GTPase Switching Mechanism Thank Autphagy Tips for proper rehydration visiting nature. You are using a Autophagy and GTPases version Auophagy limited support GTaPses CSS. To obtain the best experience, we Autophzgy you use a uAtophagy up to Recovery nutrition plan browser or turn off adn mode in Internet Peppermint candy cane. In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. Autophagy macroautophagy is a highly conserved intracellular and lysosome-dependent degradation process in which autophagic substrates are enclosed and degraded by a double-membrane vesicular structure in a continuous and dynamic vesicle transport process. The Rab protein is a small GTPase that belongs to the Ras-like GTPase superfamily and regulates the vesicle traffic process. Numerous Rab proteins have been shown to be involved in various stages of autophagy.Autophagy and GTPases -
In Sec2- and Sec4-mutant cells, Atg9 anterograde movement is impaired and Atg8 is inefficiently recruited to the phagophore membrane, resulting in reduced autophagosomes formation Geng et al. It is important to note that, in addition to its role in the expansion of the phagophore, Arf1 also regulates autophagy through the mTOR signalling pathway.
A screen of Drosophila melanogaster small GTPases found that mTORC1 activity is regulated by members of Arf and Rab GTPases, such as Arf1 and Rab5 Li et al. Upregulation of Arf1 or Rab5 strongly inhibits mTOR activity in response to amino-acid-starvation, whereas glucose-induced activation of mTOR is not affected by Arf1 and Rab5.
Moreover, Rab5, in Drosophila , selectively inhibits mTOR activation Li et al. As mentioned before, Atg9 trafficking occurs not only through the Golgi and the TGN but also through recycling endosomes.
Atg9 localisation and trafficking in this compartment is essential for the initiation and progression of autophagy Orsi et al. Rab11 is a small GTPase that localises in the pericentriolar endosomal recycling compartment and is responsible for the recycling of the transferrin receptor through that compartment Ren et al.
Rab11 was initially implicated in the regulation of autophagy Huttenhower et al. Autophagy inducers, such as rapamycin or starvation, cause indeed enlargement of Rabpositive vacuoles and a remarkable colocalisation of Rab11 with LC3 Fader et al. Subsequent studies have suggested additional roles for Rab An important functional correlation between Rab11 and the regulation of autophagy was obtained from a genetic screen of Rab GTPase activating proteins GAPs , in which several negative regulators of starvation-induced autophagy were identified Longatti et al.
The Rab GAP TBC1D14 was shown to colocalise and interact with the autophagy kinase ULK1, which, in turn, localises to recycling endosomes. Loss of Rab11 prevents TBC1Dinduced tubulation of recycling endosomes by inhibiting autophagosome formation.
Atg9 also localises in recycling endosomes together with ULK1. Taken together, these data show that Rabdependent vesicular transport from recycling endosomes contributes to and regulates autophagy Longatti et al. The exocyst, a protein complex involved in tethering transport vesicles to the plasma membrane, has been identified to act as a scaffolding complex for starvation-induced autopagy Bodemann et al.
The Ras-like small GTPase RalB, through its direct binding to the exocyst component Exo84, induces the assembly of active ULK1 and Beclin-1—Vps34 complexes at the exocyst, which is required for the isolation of the pre-autophagosomal membrane and maturation of autophagosomes Bodemann et al.
Interestingly, Vps34 activity has also been found to be regulated by Rab5, a small GTPase that is localised in the early endosomes Ravikumar et al.
Given the role of the exocyst in the transport of vesicles between the plasma membrane and the Golgi, one can hypothesise that the exocyst mediates the movement of membranes between the plasma membrane and autophagosomes. Although the most accepted role of Rab7 in autophagy refers to its ability to control the autophagosome-lysosome fusion step see below , there are a few studies suggesting that Rab7 also participates in autophagosome-like vacuole formation during the invasion of certain microorganisms, such as Streptococcus pyogenes also known as Group A streptococcus and hereafter referred to as GAS Sakurai et al.
This bacterium infects non-phagocytic cells through endocytosis, but subsequently escapes the endolysosomal pathway.
After this, GAS is engulfed by an autophagosome-like vacuole, which eventually acquires lysosomal enzymes that induce its proteolysis. The formation of a GAS-containing autophagosome-like vacuole GcAV is triggered by canonical autophagic proteins such as Atg5 Nakagawa et al.
Rab7 was found to be involved in GcAV formation and maturation, most probably by assisting in the recruitment of LC3 to GcAVs and inducing homotypic membrane fusion during the enlargement process of GcAVs Yamaguchi et al.
Rab7, indeed, appears to participate in the coalescence of multiple small Atg5-positive membranes that localise around the GAS bacteria, suggesting it has a role in the early phase of GcAV formation Sakurai et al.
In addition to Rab7, Rab9A has also been implicated in the enlargement of GcAVs and the fusion of GcAVs with lysosomes during GAS-induced autophagy. However, starvation-induced autophagy does not appear to be regulated by this small GTPase, suggesting that different types of autophagy require different factors that confer distinct specificities and functions to the different autophagy pathways Nozawa et al.
The autophagy machinery is also involved in regulating the secretion of the contents of granules or secondary lysosomes, such as Acyl-CoA-binding protein Acb1 in yeast, and interleukin 1β and interleukin 18 IL1B and IL18, respectively in mammalian cells Deretic et al.
This newly described form of secretion, which has been named autophagy-based unconventional secretion, is dependent on Rab8a, a small GTPase that is located at the Golgi and canonically involved in the polarised sorting from the Golgi to the plasma membrane.
This autophagy-dependent secretory pathway enables cytosolic proteins to exit the cell without entering the conventional ER-to-Golgi secretory pathway. It remains unclear why cells would use one particular secretion system for specific proteins instead of other systems. However, this recently unveiled aspect of autophagy may be relevant in the context of inflammatory diseases, such as cystic fibrosis and Crohn disease Cadwell et al.
Autophagy can regulate the unconventional trafficking of the cystic fibrosis transmembrane conductance regulator CFTR , the protein that is mutated in cystic fibrosis, from the ER to the plasma membrane without passing through the Golgi.
The impaired conventional Golgi-mediated secretion and cell surface expression of the CFTRΔ mutant protein can be rescued by directing it to an unconventional Golgi reassembly stacking protein GRASP -dependent secretion pathway using autophagosomes as a vehicle Gee et al.
In the context of Crohn disease, autophagy participates in the regulated secretion of lysozyme from Paneth cells Cadwell et al. With regard to bacterial infection, Rab8 has been shown to be involved in the autophagic elimination of Mycobacterium tuberculosis var.
bovis BCG through its interaction with TANK-binding kinase 1 TBK1 , which is involved in the phosphorylation of the autophagic adaptor p62 and assembly of the autophagy machinery Pilli et al.
The fusion of autophagosomes with the vacuole the lysosome in yeast is compromised in Saccharomyces cerevisiae mutants that lack the yeast Rab7 orthologue Ypt7, resulting in the accumulation of autophagosomes in the cytoplasm Kirisako et al. Many other studies that subsequently emerged aimed to characterise the mechanisms involved in the regulation of the fusion event by Rab7.
Rab7 was found to regulate the maturation of late autophagic vacuoles and the formation of the autolysosome both under nutrient replete and starvation conditions through a mechanism that is dependent on the binding and hydrolysis of GTP Gutierrez et al. More recently, a role for Rab7 has also been reported in a process named autophagic lysosomal restoration ALR , which involves the termination of autophagy and formation of nascent lysosomes from autolysosomes in a mechanism that depends on the reactivation of mTOR.
Treatment of cells with GTPγS, a non-hydrolysable analogue of GTP, completely inhibits ALR, and overexpression of constitutively active Rab7 that is permanently associated with membranes, also abrogates ALR, resulting in the accumulation of enlarged and long-lasting autolysosomes.
Inhibition of ALR by rapamycin also inhibits the dissociation of Rab7 from autolysosomes, suggesting that mTOR and Rab7 together participate in the regulation of ALR Yu et al.
Rab7- and Arl8-ancillary machinery involved in the positioning of lysosomes and autophagosomes, and their fusion. A Rab7 has been implicated in the fusion between autophagosomes and lysosomes through a mechanism that is dependent on the binding and hydrolysis of GTP.
The UVRAG—C-Vps complex appears to activate Rab7 activity and stimulate autophagosome-lysosome fusion, whereas Rubicon inhibits this step. B The movement of autophagosomes has been suggested to rely on a precise balance between dynein- and kinesin-dependent mechanisms.
Starvation conditions are likely to promote the binding of FYCO1 to kinesin, and to Rab7 and LC3-II in the membrane of autophagosomes, inducing a redistribution of the pre-autophagosomes throughout the cytosol. FYCO1 appears to compete with the RILP-dynactin-dynein complex for the binding to Rab7, providing means to regulate the bidirectional movement of autophagosomes along microtubules.
RILP-Rab7 interaction is further controlled by the UVRAG—C-Vps complex. C Left: starvation conditions, characterised by an increase of the intracellular pH, inhibit the recruitment of Arl8B which is kept on the cytosol in a GDP-bound form and of the kinesin KIF2A to the lysosomal membrane; this favours the accumulation of lysosomes and the fusion between lysosomes and autophagosomes in a perinuclear region close to the MTOC.
mTOR is also inhibited under starvation conditions, favouring the formation of new autophagosomes. Right: after nutrient replenishment characterised by a decrease of the intracellular pH , Arl8 bound to GTP is recruited to the lysosomal membrane in a complex with pleckstrin-homology-domain-containing family M member 2 PLEKHM2; here referred to as SKIP and kinesin, which binds to microtubules Korolchuk et al.
Lysosomes are subsequently transported towards the cell periphery. The centrifugal movement of lysosomes also diminishes the encounter between lysosomes and autophagosomes, which interferes with their fusion and clearance of autophagic substrates. Several groups have been focusing their efforts on the understanding of the molecular mechanisms, as well as in the identification of the molecular complexes that are involved in the regulation of autophagy by Rab7.
FYVE and coiled-coil- domain-containing protein FYCO1 has been identified in a recent study as a Rab7 effector protein that is able to bind LC3 and PtInsP 3 and mediate microtubule plus-end-directed transport of autophagic vesicles Pankiv et al. FYCO1 seems to have a role in the redistribution of Rab7- and LC3-positive vesicles to the cell periphery in a microtubule-dependent manner that might, for instance, interfere with the fusion of autophagosomes with lysosomes.
Indeed, the authors identified a potential kinesin-binding site in the central part of the coiled-coil region of FYCO1 Pankiv et al. Although the physiological implications of this mechanism were not explored in depth, the authors propose a mechanism whereby FYCO1 preferentially localises at the ER in a conformation that prevents its binding to kinesins under nutrient-rich conditions.
However, upon starvation, FYCO1 binds to the microtubule plus-end-directed motors and redistributes pre-autophagosomal membrane compartments to the sites of autophagosome formation throughout the cytosol.
It is also proposed that, after the formation of autophagosomes, FYCO1 competes with the dynein recruitment complex for binding to Rab7, providing a regulated bidirectional transport of autophagosomes along microtubule tracks Pankiv et al.
Another factor that is involved in the regulation of Rab7 is Rab7-interacting lysosomal protein RILP , a component of the complex that is responsible for the binding of Rab7 to dynactin—dynein1 Fig.
Interestingly, the interaction between Rab7 and RILP is positively affected by the activation of Rab7 through the complex of class C-Vps also known as HOPS complex — a tethering protein complex that serves multiple membrane fusion events e.
autophagosome fusion with late endosomes and lysosomes — and UVRAG, a protein known to induce autophagy and membrane curvature through a mechanism that is dependent on Beclin 1 and PtdIns 3-kinase class III Liang et al.
Indeed, the interaction between C-Vps and UVRAG stimulates Rab7 activity and promotes autophagosome maturation and fusion with lysosomes Liang et al. The GDP-GTP exchange on Rab7 necessary for its activation is likely to be dependent on the GEF activity of the C-Vps complex Liang et al.
The Rab7-RILP interaction is also regulated by the insulin-like growth factor 1 IGF1 —AKT pathway during neuronal autophagy Bains et al. An additional level of Rab7 regulation is also provided by rubicon Run domain beclininteracting and cysteine-rich-containing protein , another Beclin 1-binding protein.
Rubicon has been shown to inhibit the maturation and fusion steps during autophagy Matsunaga et al. Interestingly, rubicon appears to sequester UVRAG from the C-Vps complex and block Rab7 activation Sun et al. This response appears to be independent of a functional ER stress response pathway, because thapsigargin also blocks autophagy in ER-stress inositol requiring enzyme IRE -null cells Ganley et al.
In addition to Rab7, other trafficking-associated small GTPases probably also regulate the autophagosome-lysosome fusion step. For instance, Rab33B, a Golgi-resident Rab protein that is involved in retrograde transport, might have an indirect role in this process through the activity of its GAP OATL1 also known as TBC1D25 Itoh et al.
It has been proposed that activated Rab33B recruits the Atg12—Atg5—Atg16L1 complex to pre-autophagosomal structures, thereby inducing the subsequent conjugation of LC3 to PtdEtn.
Following this, OATL1 recognises LC3 in the autophagosomal membrane in proximity to Rab33B and inactivates Rab33B through its GAP activity, exerting a feedback loop. This mechanism appears to be involved in autophagosomal maturation and conversion of autophagosomes to autolysosomes, because inactivation of Rab33B through overexpression of OATL1 inhibits the encounter of autophagosomes and lysosomes, whereas overexpression of constitutively active Rab33B reduces the rate of fusion between autophagosomes and lysosomes Itoh et al.
Another GTPase that indirectly regulates autophagosome—lysosome fusion is the Arf-like GTPase Arl8B. A decrease of the intracellular pH — induced, for example, through nutrient-rich conditions — increases recruitment of Arl8B and the kinesin KIF2A to lysosomes, which promotes their centrifugal movement along microtubules towards the cell periphery Korolchuk et al.
This occurs concomitantly with the activation of mTORC1, which inhibits autophagosome formation and decreases autophagosome-lysosome fusion because encounters of autophagosomes and lysosomes in the perinuclear region are less likely Korolchuk et al. Activation of mTORC1 primarily occurs at the surface of lysosomes through a mechanism that depends on Rheb, ragulator and the Ras-related GTPases RagA or RagB, and RagC or RagD Sancak et al.
In this Commentary, we have described how a number of small GTPases modulate different steps of autophagy, including autophagosome formation, autophagosome secretion, autophagosome trafficking and fusion with lysosomes. For instance, Arf6 and Rab33B are small GTPases implicated in Atg16L-mediated autophagosome formation, whereas the small GTPases Rab1, Rab11 and Sec4 are likely to be involved in the formation of Atg9-autophagosome precursors.
Although the most-accepted role of Rab7 in autophagy refers to its ability to control the autophagosome—lysosome fusion step, it is also involved in the formation and maturation of autophagosomes during bacterial infection, as is the small GTPase Rab9A. In addition, RalB has been implicated in the regulation of ULK1-mediated formation of autophagosomes, whereas Rab8 controls a new and unconventional form of secretion that relies on autophagy.
The trafficking of autophagosomes and lysosomes, as well as their fusion has been shown to mainly depend on Rab7, but also requires Arl8 and Rab33B Fig. Despite extensive research efforts, the molecular networks and complexes that support the functions of these small GTPases in autophagy, as well as their spatial and temporal control, are still not completely understood and important questions still need to be answered.
In addition, it is still unclear how small GTPases reach the autophagosomal membrane. Understanding this step may shed some light into the mechanisms that underlie the intersections between endocytic, secretory and autophagic pathways.
An interesting hypothesis is that a pool of Atg9-positive membranes can cycle dynamically between the endosomal compartment, the secretory pathway and the pre-autophagosomal structures Reggiori and Tooze, ; Young et al.
However, one should not exclude the canonical process for the recruitment of small GTPases to membranes without the need of vesicular transport or fusion-dependent events, which relies on their direct recruitment from the cytoplasm to the membrane by a mechanism that is dependent on the exchange of GDP to GTP.
One can, therefore, hypothesise that the primary location of these GTPases provides clues about the membrane sources for the formation of the autophagosome. Although autophagy was considered in the past as a nonselective process, several cargo-specific autophagic processes have been recently described, including xenophagy degradation of intracellular pathogens , aggrephagy clearance of certain protein aggregates , pexophagy elimination of peroxisomes , mitophagy removal of damaged mitochondria and ribophagy elimination of ribosomes , which assist the quality control of essential cellular components reviewed by Mizushima, Autophagy occurs under basal conditions and can be induced by certain environmental stresses, such as nutrient deprivation, some infections, oxidative stress and treatment with certain drugs e.
Under starvation conditions, autophagy is induced and increases the availability of nutrients e. amino acids by releasing them from proteins and other macromolecules that are targeted for degradation.
Indeed, autophagy has roles in both health and disease conditions. It regulates early embryonic development, neonatal starvation, clearance of pathogenic bacteria during infectious processes, cancer-associated mechanisms and degradation of misfolded and aggregation-prone proteins i.
tau, mutant α-synuclein, polyglutamine-expanded huntingtin that are involved in neurodegeneration disorders, such as Alzheimer, Parkinson and Huntington diseases reviewed by Harris and Rubinsztein, ; Mizushima and Komatsu, An extensive array of signals regulates the formation of autophagosomes.
Generally, they can be categorised into mTOR-dependent or mTOR-independent stimuli. The mTOR pathway is a classic negative regulator of autophagy that is conserved from yeast to mammals. mTOR activity is inhibited under starvation conditions and rapamycin treatment, which results in the partial dephosphorylation of its targets Atg13, ULK1 and ULK2; this activates ULK1 and ULK2 to phosphorylate FIP and, thereby, induces autophagy Hosokawa et al.
In addition, mTOR is positively and negatively regulated by a plethora of other stimuli. For example, depending on the oncogenic or genotoxic stress, p53 can activate AMP-activated protein kinase AMPK , which directly activates ULK1 and also inhibits mTOR, or upregulate phosphatase and tensin homologue PTEN , which inhibits mTOR through inhibition of the Akt kinase reviewed by Ravikumar et al.
In addition, AMPK can also inhibit mTOR activity through the tuberous sclerosis complex 1 Tsc1 , Tsc2 and Ras homology enriched in brain Rheb reviewed by Ravikumar et al. mTOR can also be regulated by GTPases that influence its lysosomal localisation and activity during starvation conditions Saci et al.
Recent work has also described that the G-protein-coupled taste receptor complex T1R1—T1R3 acts as a sensor for amino acids, which then regulates mTOR activity and autophagy Wauson et al. AMPK can also regulate autophagy independently of mTOR.
Another well-characterised mTOR-independent signal that regulates autophagy includes the inhibition of inositol monophosphatase IMPase , which reduces free inositol and inositol 1,4,5 -trisphosphate [Ins 1,4,5 P 3] levels, resulting in an upregulation of autophagy Sarkar et al.
We are grateful to Fiona Menzies, Mariella Vicinanza and Maurizio Renna for critical reading of the manuscript. We thank the Wellcome Trust for a Principal Research Fellowship [to D. and a Strategic Grant to the Cambridge Institute for Medical Research.
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filter your search All content All journals Journal of Cell Science. Advanced Search. In aging, metabolic adaptation to a sedentary lifestyle could impair mitochondrial function generating less GTP and redox energy for healthy management of amyloid and tau proteostasis, synaptic function, and inflammation.
Juan A Godoy, Juvenal A Rios, … Nibaldo C Inestrosa. Kalpita Banerjee, Soumyabrata Munshi, … Gary E. ATP synthesis by oxidative phosphorylation in mitochondria is powered by NADH generation in the TCA cycle for reduction of pyruvate, glycerol, or fatty acids with oxygen as the terminal electron acceptor in the electron transport chain.
Our label-free studies in live rat or mouse hippocampal neurons indicate an age-related depletion of NAD and NADH, that could impair synthesis of ATP and GTP [ 1 , 2 , 3 , 4 ]. This depletion was further exacerbated in neurons from mice carrying transgenes for human beta amyloid and tau.
The energy available from GTP hydrolysis is the same as ATP hydrolysis, but GTP is utilized for different purposes than ATP due to the selectivity of specific enzymes.
GTP concentrations in cells are on average tenfold lower than the millimolar ATP. GTP is a major regulator of multiple energy-dependent cellular processes of protein synthesis and vesicular trafficking involving endocytosis and autophagy.
The estimated total cellular GTP concentration in mammalian cells is in the range of — μM [ 6 ], but free GTP in cancer cell protrusions is closer to 30 μM [ 7 ]. Major proteins powered by GTP include dynamins for membrane fission and fusion, the small regulatory GTPases, and microtubules as detailed below.
Beside de novo synthesis from inosine by IDMPH2 and xanthosine by GMPS, cellular sources of GTP are primarily localized nucleoside diphosphate kinases NDPKs from the non-metastatic genes NME; sometimes called NM23 that convert ATP to GTP [ 8 ]. There are at least ten members of the NME gene family but only the first four have nucleoside diphosphate kinase activity.
According to the Allen Brain Atlas, NME1 through 4 are strongly and selectively expressed in the mouse and human hippocampus where memory is impaired in AD. NME1—3 are cytoplasmic, while NME4 is mitochondrial. Since both distinct processes of endocytosis and autophagy are affected in aging and AD, we posit that alterations upstream affect both, possibly the age and AD-related consequence of limiting GTP levels on these amyloid-critical functions.
Although GTP regulates several crucial processes for cellular survival, measuring GTP levels in the live cell under physiological and pathological conditions is difficult because of high turnover and existence of both a protein-bound and free state.
Bianchi-Smiraglia et al. Recently, the same group reported a mechanism for regulation of GTPase Rac1 in cell invasion by a human melanoma cell line driven by local GTP production [ 7 ] and reviewed their methodology [ 10 ]. As we will see in this review, GTP plays a crucial role during endocytosis and autophagy related to amyloid processing.
Currently, we are studying the age-related GTP changes in processing of Aβ in primary cultures of hippocampal neurons from the triple transgenic 3xTg-AD mouse and its effect on alterations in autophagy.
Figure 1 shows preliminary results of GEVAL transfection into primary adult neurons from this AD mouse model compared to non-transgenic mouse neurons. As observed by Bianchi-Smiraglia et al. The µM Kd for GEVAL used in these experiments suggests that the local free GTP concentration is within a few-fold of µM.
In Fig. Preliminary evidence indicates that neurons treated with NME1 siRNA lowers their free GTP levels Santana Martinez, unpublished. The result of this review highlights the need for new methods like GEVAL probes to directly measure changes in bound and free GTP and total GTP concentrations.
Ratiometric GTP-measurements in primary mouse hippocampal neurons from middle-age mice transfected with GEVAL sensor False-colored pixel-by-pixel ratiometric images of neurons show non-uniform distributions of GTP. non-transgenic NTg at edges and apical dendrite compared to c middle-age 10 mo.
b , d Increase in free GTP in neurons treated with 2 mM nicotinamide by 24 h. e , g Untreated neurons exhibit vesicular bound GTP that is increased in f , h neurons treated with 2 mM nicotinamide by 24 h. In the endocytosis process, the cell internalizes macromolecules and ligand-bound receptors and surface proteins including the amyloid precursor protein APP [ 11 ] Fig.
Of the two main endocytic pathways, plasma membrane-embedded APP uptake occurs mostly through clathrin-mediated uptake Fig. However, hydrophobic Aβ, partitions into cholesterol-rich patches of lipid rafts in the plasma membrane with subsequent uptake by the caveola pathway [ 12 ] Fig. In both cases, dynamin GTPase complexes assemble at the invagination to catalyze GTP-dependent membrane curvature for final fission and release of mature vesicles from the plasma membrane [ 13 , 14 , 15 ].
Local GTP fueling is catalyzed by NME1 and 2 nucleotide phosphate kinases that bind to dynamin [ 16 ]. Endocytosis of the amyloid precursor protein APP. A The N-terminal portion of the residue APP protein with green shading of the aggregation-prone Aβ requiring cleavage by BACE and Gamma peptidases.
B Endocytosis of APP. Endocytosis supported by dynamin receives local GTP by NME1. Alternatively, 5 Rab7 mediates segregation early into late endosomes allowing further processing of APP and accumulation of Aβ. Some aggregated Aβ may be resistant to digestion, and with endosome disruption Aβ may accumulate in the cytosol.
The Rab5 GTPase is considered a marker for the early endosome [ 17 ]. Rab5 protein is present in the plasma membrane, in clathrin-coated vesicles, and early endosomes Fig. Inhibition of Rab5 reduces autophagy-regulating proteases Atg5-Atg12 conjugation, resulting in decreased autophagosome formation.
The AtgAtg5 conjugate promotes lipidation of Atg8, and lipidated Atg8 facilitates autophagosome formation and selective cargo recognition during autophagy [ 19 ]. Another contributing factor to Rab5-mediated autophagy is phosphatidylinositolkinase PI3K. The Pβ subunit of the PI3K complex regulates the catalytic activity of the Vps34 complex to promote PI3P generation [ 21 ].
This step is essential for autophagosome formation. Pβ promotes the transition from Rab5-GDP to Rab5-GTP. Pβ overexpression mitigates autophagic deficiency following activation of the macromolecular complex composed of Rab5, Vps34, and Beclin1, which in turn leads to autophagosome formation [ 22 ].
Targeting these specific proteins may elucidate therapeutic avenues to attenuate AD pathology. Membrane-bound Rab5 is a key factor to directly promote Mon1-Ccz1 dependent Rab7 activation and Rab7-dependent membrane fusion [ 23 ].
Mon1-Ccz1 is a heterodimeric guanine nucleotide exchange factor GEF-complex which activates Rab5. Mon1-mediated displacement of the Rab5 GEF results in displacement of Rab5 by Rab7. Rab7 late endocytic vesicles subsequently fuse with lysosomes for cargo degradation Fig.
In mouse models of AD with mutations in the amyloid precursor protein APP or Aβ-producing presenilin, the endosome—autophagosome—lysosome pathway appears dysregulated partly because of impaired acidification of lysosomes that fails to sufficiently activate protease and lipases [ 26 ].
In mouse models of AD, large perinuclear bodies containing Aβ aggregates label with the autophagosome marker LC3 colocalized with Rab7 and late endosomal and lysosomal components. This could be due to failed protective upregulation or pathologic impairment. In neurons cultured from 3xTg-AD mice across the age-span, we found aggregated vesicular Aβ to increase 30—50 fold with age, most prominently in Rab5-labeled early endosomes and mitochondria, but also within Rab7-labeled late endosomes and autophagosomes [ 27 ].
The snow-ball-like accumulation of Aβ42 and Aβ45 suggests that old neurons were unable to complete autophagic degradation of these longer aggregates of Aβ. We hypothesize that impaired energy production in old neurons limits the energetic capacity for completion of autophagy. Rab7 deficiency in yeast and fruit flies results in massive accumulation of autophagosomes [ 29 ].
Interestingly, the inhibition of other stages of endocytic trafficking does not change the activity of mTORC1 suggesting that intact late endosomes are crucial for mTORC1 signaling in autophagy.
This is an important reason why targeting mTOR may not slow aging or AD. Regionally selective upregulation of Rab 5 and Rab7 proteins and mRNAs in AD, suggests selective contributions to disease pathology or an insufficient protective mechanism [ 31 ].
However, practical interventions to promote Rab7 mediated autophagy could delay or reverse AD progression. Examples include a shift from a sedentary state to exercise, Mediterranean diet, and energy boosting compounds like NAD precursors to create a redox shift [ 4 ] Section Another avenue is the strange discovery that guanosine monophosphate reductase 1 NADPH-dependent GMPR1 levels are increased in AD brains that could itself lower GTP levels and raise AMP signaling [ 37 ].
Neurofibrillary tangles of tau were lowered when this activity was inhibited in AD-mice. With progression from mild cognitive impairment to AD, both endosomal Rab5 and Rab7 expression were upregulated in hippocampal CA1 neurons as measured by transcriptional microarray [ 31 ]. Enlargement of Rab5-positive endosomes was associated with neurofibrillary tangles and amyloid deposition.
Neurotrophic factors such as nerve growth factor NGF bind to their Trk receptors and internalize into Rab5-positive endosomes to initiate downstream signaling [ 38 ]. It is interesting to note that, the signaling endosome is retained as Rab5 early endosomes, and does not progress to Rab7 late endosomes, during their transit within the long axons [ 39 ].
This differentiates receptor-mediated signal delivery from endosome processing toward autophagy [ 40 ]. Rab5 function is compromised in early phases of AD [ 41 ]. Persistent hyperactivation of Rab5 promoted the endocytic pathway toward late endosomes, lysosome fusion, and autophagy resulting in premature degradation of the neurotrophic factor signaling and neuronal atrophy.
Vps35 and Vps26, two key retromer proteins, were also reduced in AD brains [ 42 ]. Vps26 binds to SorLA which is a sorting receptor that controls APP trafficking from endosomes to the Golgi. A reduction in Vps retromer proteins leads to abnormal Vps-SorLA complex that hinders APP trafficking casing APP accumulation in the endosomes where it is subject to beta-secretase.
However, mimicking APP phosphorylation at S, within the APP YTSI basolateral motif, can enhance APP retrieval in a retromer-mediated process causing decreased APP lysosomal targeting, and decreased Abeta production [ 43 ].
The net result with aging is depletion of late endosomes, late endocytic dysfunciton, and impaired lysosomal fusion. In AD, endocytosis of Aβ increases into enlarged Rab5 early endosomes.
The role of Rab7 in AD is yet to be clearly elucidated as both early upregulation and late downregulation of Rab7 have been observed affecting neuronal health in both directions. As emphasized by Bianchi-Smiraglia et al.
Free GTP measures have not been reported yet, so their possible role in age and AD-related impairments in endocytosis is unexplored. Therefore, it will be important to measure free GTP in live cells and the effects of variations in free GTP on endocytosis as a function of age and in AD models.
The GTPase superfamily comprises a wide range of proteins that act as molecular switches by binding and hydrolyzing GTP molecules for tethering, docking, and fusion of vesicles to target membranes [ 46 ]. Changes in GTPase function are related to alterations in the trafficking of cargo [ 47 ].
For many GTPases, the lifetime of the activated, GTP-bound state is believed to serve as a regulator in determining the activation time of a biological event such as membrane fusion and signal transduction. However, proper function of the GTPases could also be limited by a decrease in intracellular GTP levels.
Molecular switch between GTPase states. Active GTPase interacts with several kinds of downstream effectors to modulate their activity. GTPase activating protein GAP inactivates the GTP-bound proteins by boosting their activity for GTP hydrolysis.
The GDP-bound form cannot bind effectors. Autophagosome formation involves Rab family members Rab1, Rab5, Rab7, Rab9A, Rab11, Rab23, Rab32, and Rab33B. Rab9 is required in non-canonical autophagy. Rab7, Rab8B, and Rab24 have a key role in autophagosome maturation. Rab8A and Rab25 are involved in unknown aspects of autophagy [ 22 ].
Rab11 is required for exocytosis [ 49 ]. Rab8b has a key role in orchestrating autophagic maturation although its downstream effector Tbk-1, which directly phosphorylates p62 at Ser, a crucial residue for autophagic function of p62 [ 50 ].
TBK-1 is also required for cytokinesis-induced autophagic elimination of bacteria, whereas Rab8 knockdown after induction of autophagy caused a decrease of phagosomes [ 50 ]. Rab GAPs facilitate the hydrolysis of GTP. Both regulators are required to coordinate the temporal-spatial activity of Rab GTPases [ 52 ].
The activity of Rab GTPases, GEFs, and GAPs are crucial for transport and trafficking of autophagosomes for macroautophagy. Macroautophagy needs stringent control, and some Rab GAPs seem to function in overlapping pathways.
For example, TBC domain containing proteins, TBC1D14 and TBC1D15, coordinate endosomal trafficking and autophagosome biogenesis. TBC1D5 is a Rab7 GAP that is recruited to mitochondria by protein FIS1 to handle Rab7 GTP hydrolysis, allowing also mitochondria to regulate the contact untethering with lysosomes [ 53 ].
Since mitochondria-lysosome contacts mark the sites of Drp1-positive mitochondrial fission events, alterations in the Rab7 GTP hydrolysis lead to both abnormal lysosomal morphology and markedly reduced the rate of mitochondrial motility [ 53 ].
TBC1D2, which affects the Rab7 GTPase and modulates autophagosome-lysosome fusion, was shown to be activated by LRRK1 upon macroautophagy induction [ 54 ]. The protein family of small Rab GTPases control vesicle transport routes and ensures trafficking of vesicles to their appropriate target compartments.
Rab GTPases interact with effector proteins such as cargo sorting complexes, motor proteins, and tethering factors, which are required for vesicle budding, transport, and fusion of various intracellular organelles. In mammalian cells, there are three primary types of autophagy: macroautophagy, microautophagy, and chaperone-mediated autophagy CMA Fig.
Each subtype consists of different mechanisms of substrate delivery to the lysosome; however, the result is the same for all of them culminating in the delivery of cargo to the lysosome for degradation and recycling. Types of autophagy pathways regulated by GTP A In macroautophagy mitophagy , ubiquitin Ub -labeled proteins recruit p62 to interact with LC3 to form autophagosomes.
Rab2 participates in phagophore formation, whereas Rab8b and Rab9a participate in autophagosome maturation. Arl8 is located on the lysosome membrane and facilities lysosomal trafficking. B In chaperone-mediated autophagy CMA , substrate proteins bind to the monomeric form of LAMP-2A after recognition by a KFERQ motif by cytosolic Hsc70 chaperon complex.
Unfolding of the complete substrate is required for its translocation into a multimeric complex with LAMP-2A. There are two pools of GFAP in the membrane of lysosomes: 1, in conditions with high CMA activity, GFAP interacts with LAMP-2A to stabilize the complex required for the translocation of CMA cargo in a GTP-dependent manner; 2, GFAP interacts with EF1α, while GTP induces the release of EF1α from GFAP inhibiting CMA and promoting the disassembly of multimeric LAMP-2A.
In microautophagy, autophagic cargo are engulfed by invaginations of the lysosomal membrane to capture content. In all of them, recycling occurs after lysosomal degradation. Macroautophagy of mitochondria is the most studied and is commonly referred as mitophagy.
Mitophagy signaling is mainly controlled by the Target of Rapamycin protein complex 1 TORC1. Following induction of autophagy, a sequestering membrane called a phagophore is formed Fig. The autophagosome then fuses with the lysosome, transferring the cytoplasmic cargo for hydrolysis.
CMA Fig. Reactive oxygen species ROS partially controls autophagy. Starvation stimulates ROS mainly H 2 O 2 production in mitochondria, which appears to be necessary for autophagosome formation. In yeast, autophagy can be regulated by ROS via Atg4, through oxidation—reduction of a disulfide bond between residues Cys and Cys , which is required for a proper autophagosome biogenesis [ 58 ].
Atg4, a redox protease, acts as a conjugating enzyme cleavage C-terminus in immature Atg8 mammalian homologue LC3 to expose the conserved glycine residue for its subsequent linkage to phosphatidylethanolamine PE.
Further, Atg4 acts also as a deconjugation enzyme that cleaves the amide bound between Atg8 and PE, which releases it from the membrane for recycling, which is crucial for the conjugation systems essential for autophagy.
Autophagic clearance of damaged cellular components or aberrant protein aggregates becomes increasingly important to handle the increased oxidative stress associated with aging and AD [ 59 ]. The bidirectional trafficking into and out of cells must be highly coordinated. Autophagy works in association with endocytosis and exocytosis, both of which contribute to turnover of damaged intracellular constituents employing lysosomal fusion and digestion.
Autophagy involves tagging defective cellular organelles and protein aggregates for degradation, followed by the assembly of an autophagic structure to transport cargo for fusion with lysosomes to degrade damaged proteins and organelles for recycling amino acids and lipids or disposal.
It is a highly dynamic process essential to maintain cellular homeostasis and functions. Although it is well known that ATP levels decline with age under pathological conditions [ 60 ], age-related changes in GTP levels are poorly elucidated.
Decreased ATP production stimulates AMP-activated protein kinase AMPK , and stimulation of AMPK inactivates mTOR. AMPK increases autophagy not only indirectly through inactivation of mTOR but also directly through phosphorylation of Unclike kinase 1 Ulk1 which is the molecular target of mTOR in the autophagic machinery [ 61 ].
Post-mortem analysis of AD patients indicates an accumulation of autophagosomes and other prelysosomal autophagic vacuoles in dystrophic neurites and synaptic terminals, which are neuropathological hallmarks of AD [ 62 ].
These facts suggest that AD is associated with alterations in trafficking of autophagosomes. Autophagy dysregulation occurs in both AD patients and animal models. Similarly, immature autophagic vesicles in axons were observed in hippocampal neurons of AD mice, far before synaptic and neuronal loss Cataldo et al.
Tau aggregates are also degraded through the autophagy pathway [ 67 , 68 ]. The wild type presenilin gene 1 PS1 acts as a ligand of the v-ATPase V0a1 subunit regulating the distribution of v-ATPase subunits into lysosomes for acidification.
Intracellular Aβ binds to v-ATPase and inhibits acidification [ 69 ]. Mutation in PS1 thus contributes to the dysregulation of the autophagy-lysosome degradation system [ 70 ].
The major genetic risk factor for sporadic AD, Apolipoprotein E4 ApoE4 , also contributes to induction of autophagy by lysosomal leakage [ 71 ] leading to impaired endolysosomal trafficking, disruption of synaptic homeostasis and reduced amyloid clearance.
Altogether this suggests that the defective autophagy-lysosome proteolysis pathway might be responsible for the accumulation of pathogenic proteins such as Aβ and tau in AD. This observation suggests that alterations in autophagosome formation avoids proper Aβ processing that could lead an aberrant accumulation in the soma [ 27 ].
Aβ accumulation has been also observed in the cis- and trans-face of the Golgi vesicles in the late Golgi apparatus, indicating this organelle could also produce functional alterations that impair the proper formation of the phagophore [ 73 ]. Supporting this supposition, the small GTPase Rab2 connects the Golgi network to the autophagy pathway machinery [ 74 ].
Rab2 participates in the formation of phagophores by further recruiting and activating Ulk1. Rab2 interacts with Rubcnl and Stx17 an autophagosomal SNARE protein to further specify the recruitment of HOPS complex to facilitate autophagosome maturation and fusion with lysosomes [ 74 ].
Another member of Ras super family of small GTPases involved in vesicle formation is the ADP-ribosylation factor Arf. Arf GTPase mainly participates in the budding process in the Golgi complex, recruitment of coat proteins during vesicle formation for membrane trafficking.
Arf GTPase is involved in control of APP trafficking through MINTs proteins, crucial components for the fusion of synaptic vesicles.
The knockdown of Arf1 decreased secretion of amyloid peptides [ 76 ], suggesting the impact of failures in Arf1 function on trafficking of APP, which could converge in intracellular accumulation Fig.
Autophagy requires the participation of several small GTPases. In the initiation phase, Rab2 recruits and activates ULK1 in the phagophore formation, while Rab9a and Rab8b participate in autophagosome maturation.
Arf takes part in budding in the Golgi complex, and recruitment of coat proteins during vesicle formation. Rab1 participates in the bidirectional vesicular transport route between the endoplasmic reticulum ER and Golgi apparatus.
Sar1 GTPase is involved in the COPII-mediated transport as the exit route of the APP once its final conformation has been reached.
Rab6 resides in the TGN and participates in retrograde transport from Golgi to ER, and has been associated with the regulation of the vesicular transport and processing of APP.
Rab11 controls the endosome recycling to the plasma membrane. Disruptions in vesicular export that contains APP-BACE could cause an accumulation of exosomes. Arl8 regulates the transport and lysosomal fusion via microtubules along the neuron.
Dysregulation of various Rab proteins also contributes to AD pathology. A defect in Rab6 can influence the secretion of APP into the medium, leading to alterations in the secretion rate, which could promote changes in the anterograde trafficking of APP leading to intracellular accumulation.
These data suggest that the accumulation of Aβ could also be generated by failures in the export of APP-containing vesicles, which could give way to a cleavage by the secretase in the vesicular membrane [ 27 ] Fig.
Since formation of the APP-containing vesicles and the formation of the phagophore involve a correct functioning of the ER-Golgi driven by small GTPases, deficits in GTP levels would cause disturbances in the packaging of APP that could compromise its secretion and promote intracellular accumulation.
These relationships of autophagy to accumulation of intracellular Aβ are supported by an age-related colocalization signal between pdirected formation of autophagosome and Aβ forms observed in primary cultured hippocampal neurons from adult 3xTg-AD mice [ 27 ] and in APPtransgenic mice with Atg7 floxed mice [ 73 ].
Another interesting fact is that no signal of aggregates was observed in autophagolysosomes positive for cathepsin D [ 27 ].
These data may indicate dysfunction in earlier steps in autophagy or in lysosomal function in AD model neurons Fig. Deficiency in autophagolysosome formation in AD. Mature autophagosomes p62 and LC3 positive cells containing Aβ aggregates are accumulated in the soma and axons leading to vesicular imbalance further associated with energy failure.
Instead of organelles, CMA degrades small molecules in eukaryotic cells induced by prolonged starvation or mild oxidative stress. CMA has been proposed to be regulated by GTP levels [ 80 ]. This regulation involves the participation of the intermediate filament glial fibrillary acidic protein GFAP and the elongation factor-1 alpha EF1α [ 46 , 81 ].
GFAP is present in two different pools at the lysosomal membrane, a portion bound to LAMP-2A and another unbound form that interacts with EF1α. The three KFERQ motifs in the Hsc70 chaperon direct it to lysosomal membranes where it interacts with protein complex LAMP-2A and stabilizes the translocation of CMA-cargo into the lysosomal lumen.
The portion of GFAP not bound to LAMP-2A contributes to GTP regulation. In the presence of GTP, EF1α is released from GFAP at the lysosomal membrane which promotes the dissociation between GFAP and LAMP-2A, mobilizing LAMP-2A to the lipid microdomains for its degradation and subsequent CMA inhibition [ 81 ].
Failures in LAMP2A complex density on lysosomal membrane are also associated with aging, which also leads to a decrease in CMA function [ 82 ] Fig. Yet another form of mammalian autophagosome biogenesis operates through an enigmatic non-canonical VPSindependet pathway.
Phosphoinositides PIs define the membrane identity and control several membrane trafficking events. Phosphatidylinositol 5-kinase PIKfyve converts endosome-localized phosphatidylinositolphosphate PI 3 P to PI 3,5 P2, a key regulator of early to late endosome membrane trafficking [ 83 ].
Further, PIKfyve complex is also responsible for production of PI 5 P from PIs and regulates autophagosome formation. PI 5 P regulates autophagy via PI 3 P effectors recruiting WIPI2 and DFCP1 proteins , which provide a mechanistic framework for this alternative autophagy pathway.
PI 5 P is used by phosphatidylinositol 5-phosphate 4-kinase β PI5P4Kβ that regulates PI 5 P levels using GTP rather than ATP for PI 5 P phosphorylation to obtain PI 4,5 P2 as a final product that regulates actin cytoskeleton remodeling [ 84 ].
PI5P4Kβ activity is proposed to reflect changes in direct proportion to physiological GTP concentration, acting as an intracellular GTP-sensor [ 85 ]. In cells lacking PI3P low PI 3,5 P2 with locked VPS34, the PIKfyve complex sustains autophagy through the use of PI5P [ 85 ].
These data indicate PIKfyve has a pivotal role in the modulation of autophagy. Impaired PIKfyve function drives formation of swollen vacuoles, easily visible at low magnification in living cells [ 86 ].
The intracellular domain of APP binds the Vac14 subunit of PIKfyve complex, affecting PI 3,5 P2 production [ 87 ]. PI 3,5 P2 binds and activates the endolysosomal TRPML channel [ 88 ]. They found enlarged vacuoles in PI 3,5 P2-deficient mouse fibroblasts that were suppressed by overexpression of healthy TRPML1 channel.
TRPML conductivity and lysosomal acidification were impaired [ 89 ]. In addition to providing ATP through oxidative phosphorylation, mitochondria also provide GTP from NME4 and its nucleoside diphosphate kinase activity [ 8 ].
This mitochondrial supply of energy is essential for generation of synaptic vesicles for release at axon terminals as well as for vesicular recycling at synapses. Synaptic loss in AD could be caused by loss of bioenergetic capacity to maintain these essential processes.
Therefore, the number and localization of mitochondria to synapses likely determines the energetic capacity for endocytosis and exocytosis.
Mitochondrial dynamics delicately balance fission and fusion controlled by Drp1 and Fis1, Mf1, Mfn2 and Opa1 [ 90 , 91 ] Fig. ATP conversion to GTP is locally controlled by localized nucleoside diphosphate kinases NDPKs from the NME genes 1—4 [ 51 ].
NME1 and 2 sometimes called NM23 H1 and H2 are predominantly cytosolic, while NME3 and 4 NM23 H3, H4 are mitochondrial. The mitochondrial NDPKs complex with specific dynamin GTPases to channel GTP directly from ATP hydrolysis [ 16 ]. The balance of mitochondria fission and fusion is sensitive to redox imbalance.
Either endogenous or exogenous application of ROS activates mitochondrial fission, inducing mitochondrial fragmentation and subsequent mitochondrial dysfunction [ 90 ]. This leads to further ROS overproduction and a vicious cycle that amplifies oxidative stress and ultimately causes oxidative imbalance in AD [ 92 ].
They polymerize and constrict tubular membranes much like endocytosis. Fis1 is localized in the outer mitochondrial membrane [ 90 , 93 , 94 ].
GTP-dependent mitochondrial dynamic morphology of fission and fusion. Left fission panel: Dynamin-related protein-1 Drp1 executes the mitochondrial fission by self-polymerizing around the outer mitochondrial membrane constricting and severing both membranes in a process dependent on GTP hydrolysis.
OPA1 enables inner mitochondria membrane fusion using local GTP provided by NME4. Red dashed arrows indicate the displacement of the organelle. Fusion is controlled by three GTPase proteins: Opa1, located in the inner mitochondria membrane and Mfn1 and Mfn2, located in the outer mitochondrial membrane.
The Opa1 GTPase with a Km around μM would require abundant levels of GTP to polymerize and mechanize mitochondrial membrane fusion into tubes [ 51 ]. Mitophagy can be activated by several stimuli such as hypoxia, energetic stress, and increased oxidized redox state.
Increased oxidative stress and elevated ROS levels caused fragmentation of mitochondria and induction of DRP1 fission-dependent mitophagy in mouse and HeLa cells.
This did not result in cell death and autophagy because moderate levels of ROS were not sufficient to trigger non-selective autophagy [ 95 ]. Indeed, this mitophagy can be inhibited by N-acetyl-l-cysteine through fueling of the glutathione pool and possible action on Atg4.
A decrease of the glutathione pool also induced mitophagy but not general autophagy. Conversely, the addition of a cell-permeable form of glutathione inhibited mitophagy [ 96 ].
Thus, an oxidative redox state promotes target-selective removal of dysfunctional mitochondria. This also suggests integration of redox balance and energy levels to maximize healthy mitochondrial function and control turnover of damaged mitochondria.
Impairment in fusion and fission has been implicated in AD. In a mouse model, Aβ interacts with fission protein Drp1, with a subsequent increase in free radical production, which further activates Drp1 and Fis1, causing excessive mitochondria fragmentation, defective transport of mitochondria to synapses, lowers synaptic ATP, and ultimately leads to synaptic dysfunction [ 97 ].
p-tau also interacts with Drp1 and enhances GTPase Drp1 enzymatic activity, leading to excessive fragmentation of mitochondria and mitochondrial dysfunction in AD [ 98 ]. A Drp1 S-nitrosylation adduct SNO-Drp1 , further stimulated its activity, and led to excessive mitochondrial fragmentation, and synapse loss [ 99 ].
Lysosomal digestion of autophagic cargo is the last step for the completion of autophagy. Therefore, lysosomes must maintain its acidic milieu for pH-based degradation of cargo by acid-activated peptidases, lipases, nucleases, and glycosidases.
For lysosomal acidification, the influx of protons is carried out by both the v-ATPase, an ATP-dependent proton pump and chloride proton antiporters, while cation efflux is mediated by transporters TPC and TRPML, which are also involved in the pH balance [ ]. Presenilin-1 PS1 regulates the distribution of v-ATPase subunits into lysosomes acting as a ligand of the v-ATPase V0a1 subunit and maintains lysosomal homeostasis via TRPML1 [ 69 ].
PS1 mutations have been linked to low lysosomal acidification, dysregulation of the autophagy-lysosome degradation system and the pathogenesis of early onset AD [ 26 ].
Arl8 an Arf-like G protein is a small GTPase located on lysosomes that acts as a linker between lysosomes and kinesin-1 to facilitate lysosomal trafficking along axons Fig. Arl8b also acts as a switch to regulate the association of HOPS complex with the lysosomal membrane [ ].
Disruption of Arl8b function causes abnormal accumulation of cholesterol in the membranes of lysosomes driving impaired axonal lysosome trafficking and leading to autophagic stress and axonal autophagosome accumulation [ ]. Additionally, elevated Arl8b expression rescued lysosome transport into axons and autophagic stress.
Overexpression of Arl8 also stimulated the bidirectional motility of lysosomes on microtubules by binding to the kinesin-1 linker SKIP to link kinesin and power motility [ ]. SKIP required Arl8 in its active GTP-bound state for binding.
However, overexpression of Arl8b also caused a striking alkalinization of lysosomes and movement toward the cell periphery versus a more uniform distribution of lysosomes throughout the cytoplasm [ ].
A proteomic study in human tissue reported enrichment of Arl8b in amyloid plaques [ ]. These data suggest that changes in the expression or functioning of Arl8 affects the fusion of the lysosome with the autophagosome or late endosome, which could have implications in autophagosome accumulation observed in AD.
Since formation of secretory vesicles delivered to the plasma membrane is a GTP dependent process, requiring Arf and Rab GTPases, deficits in GTP levels may compromise proper vesicular trafficking. Dynamic instability of microtubules associated with GTP. Heterodimers are added to the growing microtubule lattice for polymerization forming a new layer of GTP-heterodimers known as a GTP-cap.
Depolymerization occurs when heterodimers leave the shrinking microtubules lattice. Hyperphosphorylation of microtubule associated protein, tau promotes neurofibrillary tangle NFT formation and microtubule destabilization. The transition from a growing state to a catastrophic shrinking state.
B Endolysosome formation requires lysosomes move along microtubule tracks in the positive direction, while late endosomes move in the negative direction. The lysosomal multiprotein complex BORC not shown activates the small GTPase Arl8 to engage kinesin-driven plus-end transport.
For minus-end transport, the Rab7-GTP-bound state recruits the Rab-interacting lysosomal protein RILP and the cytosolic oxysterol-binding protein-related protein 1 ORP1L forming the dynein-dynactin complex. NFT formation disrupts vesicular traffic along microtubules.
Lysosome biogenesis is regulated by mTORC1 and the transcription factor EB TFEB creating a reversible signaling complex on the lysosome surface. mTORC1 phosphorylates TFEB on Ser, driving thereby enabling TFEB transport to the nucleus to upregulate v-ATPase expression and other genes involved in lysosome biogenesis and autophagosome formation [ , ].
Addition of Aβ to microglial cells lowers TFEB in the nucleus and impairs processing of Aβ [ ]. This regulation by mTORC1 is in turn supported by small GTPases including Rheb and Rag, amino acid-sensing components included in the multiprotein signaling complex regulator which acts as an activator of mTORC1 [ ].
Since lysosomal acidification requires ATP for vATPase function and GTP for GTPase-mediated regulation of TORC1, we hypothesize that energy depletion due to aging or AD-like pathological conditions directly impairs lysosomal function. Additionally, considering that lysosomal acidification involves a nutrient intake-mediated regulatory interaction between v-ATPase and TORC1 via TFEB, cycles of fasting and nutrient consumption could benefit lysosomal function in AD [ ], while frequent sugar consumption may impair function.
Impaired lysosomes could lead to accumulation of autophagosomes full of damaged mitochondria and enriched with Aβ-aggregates incapable of being degraded. Since autophagy carries out the replacement of damaged or aged organelles, its maintenance is affected by metabolic changes due to age. These observations strongly suggest a relationship between alterations in the maturation process of the autophagosome and the metabolic deficiencies that occur with the progression of the disease and ageing.
Further, aberrant Aβ accumulation could be the consequence of upstream deficiencies in GTP that impair autophagic processing of Aβ and tau, possibly earlier than aggregate amyloid secretion, inflammation and plaque buildup. Microtubules are key players in axonal growth and provide structural support to axo-dendritic vesicular trafficking.
Tubulin heterodimers bind to two molecules of GTP at two separate sites. The N-site is located at the intradimer interface, between α- and β-tubulin at which GTP is not hydrolyzed and exchanges at a slow rate. The E-site is at the intradimer interface formed by the β-subunit of one heterodimer and the α-subunit of a neighboring heterodimer.
GTP at the E-site is hydrolyzed to GDP and exchanged for a new GTP nucleotide. Since microtubules exhibit a high rate of dynamic equilibrium between polymerization states, cytoplasmic levels of GTP must be high enough to power the demand of the dynamic instability in the microtubes Fig.
Microtubules form the cytoskeletal tracks on which lysosomes travel to their target endosomes and phagosomes Fig. The kinesin motor powers transport of the lysosomes toward the positive-end of axonal microtubules with ATP, but the connection of the kinesin motor to the lysosome requires Arl8 GTPase with bound GTP [ ].
Conversely, transport of late endosomes on microtubules toward lysosomes requires Rab7 GTPase and bound GTP to attach the dynein motor.
In AD, the microtubule-associated protein tau polymerizes with hyperphosphorylation into insoluble filaments of axo-dendritic neurofibrillary tangles NFT [ , ]. Once hyperphosphorylated, tau loses it affinity for the microtubules.
Wide-ranging studies have approached the alterations in microtubule dynamics, but have failed to identify a clear cause of failure in tau function. Regulation of tau involves GTPases. In vitro, Rac1 GTPase activation caused hyperphosphorylation of tau , increased Abeta42 production and decreased actin stability in spines [ ].
This group also saw a biphasic rise in Rac1 GTPase in young 3xTg-AD hippocampus, followed by a later decline, as in late-stage human AD brains. In another study of tauopathy mice, increased farnesyl transferase activity to translocate the Rhes GTPase to autophagic vesicles was associated with low tau hyperphosphorylation, while inhibition of Rhes promoted tau hyperphosphorylation [ ].
Thus, GTPase-dependent autophagy effectively clears pTau [ ] and impaired autophagy from low GTP levels or oxidative redox state would impair clearance of pTau.
GTP is also essential for protein synthesis, the synthesis side of proteostasis, that requires the hydrolysis of two GTP molecules for each amino acid incorporated into a polypeptide. While ATP is used for charging aminoacyl-tRNA, for RNA helicase and recycling GDP to GTP, GTP itself is essential for the activity of elongation factors at intitiation IF-2 , elongation EF-Tu, EF-G and ribosome release factor for termination RF1 [ ].
This group used a cell-free reconstituted system to measure an overall Km for ATP of 27 µM and 14 µM for GTP. Consequently, a large amount of GTP is required at synaptic plasticity where metabolic demands are the highest to maintain ionic homeostasis for synaptic function and protein turnover.
Protein synthesis is crucial for presynaptic neurotransmitter release as well as consolidation of post synaptic plasticity [ ]. Nevertheless, protein synthesis was decreased along with ribosomal RNA and tRNA levels, while RNA oxidation increased in the early phase of AD [ ].
Altered protein synthesis leads to a constant accumulation of oxidized proteins leading to misfolding and aggregation. Protein aggregation in turn impairs the activity of cellular proteolytic systems resulting in further accumulation of oxidized proteins [ ].
This protein promotes the formation of clusters of vesicles, and a membrane sometimes surrounds these clusters. Further experiments indicate that several proteins involved in a process called autophagy—where unwanted proteins and debris are destroyed—may also be found around the clusters of vesicles.
Autophagy starts with the formation of a membrane around the material that needs to be destroyed. This seals the material off from rest of the cell so that enzymes can safely break it down.
Binotti, Pavlos et al. These findings suggest that excess vesicles at synapses may be destroyed by autophagy. Further work will be required to establish how this process is controlled and how it is involved in the loss of synapses.
Synapses are highly dynamic structures exhibiting frequent turnover. The most dramatic phase of synaptic remodeling occurs during development when the majority of initially formed synapses are eliminated while the final synaptic network is being generated. However, even in the adult brain there is persistent turnover of synapses, mostly in response to experience and learning Caroni et al.
Formation of a new synapse involves the establishment of highly specialized structures containing arrays of unique membrane and scaffold proteins, which necessitates close coordination between the presynaptic axon and the postsynaptic dendrite.
Components of these structures are delivered by microtubule-based transport, although some of the proteins are locally synthesized in dendrites Steward and Levy, ; Holt and Schuman, Similarly, delivery of synaptic membranes such as synaptic vesicles and active zone precursors to the synapse relies on kinesin-mediated transport Hirokawa et al.
A lot has been learned in recent years about the signaling events and the downstream effectors involved in synaptogenesis as well as the mechanisms by which individual components are recruited and maintained Caroni et al. Less is known, however, about the molecular cascades involved in synaptic elimination.
Elimination from the outside is usually executed by microglial cells but the underlying signaling network is complex, and astrocytes have also recently been appreciated to play a major role in this process Chung and Barres, ; Stephan et al.
In cell autonomous elimination, synaptic components may either be i recycled, that is, being removed in a functionally intact form for the use at another site, or ii degraded. With the exception of some recent evidence showing that synaptic vesicles can be exchanged between neighboring synapses, whether synaptic components can be reused after having been operational in a functional synapse remains largely unexplored Darcy et al.
Conversely, an increasing body of evidence supports the view that synaptic components are rapidly degraded once a synapse is earmarked for elimination. Unsurprisingly, the ubiquitin-proteasome system is emerging as one of the central players, both at presynaptic Yao et al.
At the presynaptic site, the ubiquitin system is not only involved in synaptic elimination, but also in the general regulation of synaptic plasticity Muralidhar and Thomas, ; Campbell and Holt, ; DiAntonio et al. For instance the protein RIM, a crucial hub for organizing active zones that form the release site for synaptic vesicles, was recently shown to be rapidly turned over upon ubiquitination, resulting in loss of synaptic function Yao et al.
Furthermore, an increasing number of ubiquitin-modifying enzymes have been described from synapses particularly E3-ligases Ding and Shen, In contrast to the emerging role of the ubiquitin proteasome system, little information is currently available regarding the mechanisms by which synaptic membrane proteins are eliminated.
At the postsynaptic site, ubiquitin-dependent pathways are clearly involved in the regulation of surface receptor density Patrick et al. However, only scant information is available about the turnover of membrane proteins at the presynaptic site where a complicated and autonomous vesicle recycling machinery needs to deal with many s of synaptic vesicles.
Surprisingly, the mechanisms by which synaptic vesicles are eliminated have thus far received little attention. By analogy to non-neuronal cells, it is frequently assumed that synaptic vesicle membrane proteins follow the canonical endosomal-lysosomal route for degradation which involves ubiquitination and recognition by the ESCRT machinery after being delivered to endosomes, followed by the formation of multivesicular bodies, retrograde transport, and ultimately fusion with lysosomes Katzmann et al.
However, aside from a few hints from a recent proteomic study, whether synaptic vesicle proteins are ubiquitinated remains unclear Na et al.
Similarly, whether sequestration into the lumen of multivesicular bodies is involved and, if so, to what extent is unknown. Indeed, multivesicular bodies are infrequently observed in axons and typically appear in response to pathological dystrophic or toxic conditions for review see [ Von Bartheld and Altick, ].
Furthermore, no information is currently available concerning the involvement of the ESCRT pathway in the elimination of presynaptic components. Intriguingly, recent studies implicate the involvement of clathrin-dependent pathways in targeting plasma membrane components to autophagosomes, hinting at the potential involvement of this mechanism in the turnover of synaptic vesicles recovered by endocytosis following the release of their neurotransmitter content Ravikumar et al.
In this study we report about data suggesting the presence of a novel pathway for the degradation of synaptic and secretory vesicles, which involves selective sequestration of vesicle clusters into structures resembling early autophagosomes.
We show that Rab26 selectively localizes to presynaptic membrane vesicles and recruits both Atg16L1 and Rab33B, two components of the pre-autophagosomal machinery. Remarkably, these autophagosomal structures are filled almost exclusively with synaptic vesicles and proteins typically associated with large dense-core vesicles.
Overexpression of EGFP-tagged Rab26, but not of FLAG-tagged or wild-type WT Rab26, induces the formation of giant autophagosomes in the cell bodies of hippocampal neurons—a phenotype that is mirrored upon transfection in HeLa cells.
Based on these findings, we conclude that Rab26 may selectively channel synaptic vesicles into pre-autophagosomes and, thus, may represent a new regulator of synapse turnover.
Previously we reported that synaptic vesicles highly purified from rat brain contain more than 30 different Rab-GTPases Takamori et al.
Of these, a subgroup of Rabs including Rab3a, Rab3b, Rab3c, and Rab27b were highly enriched in the vesicle fraction Pavlos et al. Rab26, a comparatively uncharacterized member of the Rab superfamily, is also closely related to this subgroup.
Since we detected Rab26 on purified synaptic vesicles in two previous independent proteomic studies Takamori et al. To this end, we raised a mouse monoclonal antibody that is specific for Rab26 and does not cross-react with other related Rab proteins including Rab27 Figure 1—figure supplement 1.
First, we used immunoblotting to monitor the subcellular distribution of Rab26 during the purification of synaptic vesicles from the rat brain. As shown in Figure 1A , Rab26 co-purified with synaptic vesicle markers as indicated here using synaptophysin , with the highest enrichment being observed in the synaptic vesicle SV fraction obtained after purification using consecutive density gradients and size exclusion chromatography.
For independent confirmation, we carried out immunoisolation of synaptic vesicles using beads Eupergit C1Z covalently coupled with monoclonal antibodies specific for Rab26 or synaptophysin.
As shown in Figure 1B , both antibodies resulted in the isolation of membranes highly enriched in both synaptophysin and Rab As a control, the membranes were solubilized with the detergent Triton X prior to immunoisolation Tx-IP. In this case, only the respective antigens were isolated Figure 1B , thus validating the specificity of the isolation procedure.
We also verified the nature of the immunoisolated vesicles by transmission electron microscopy TEM. As previously reported, synaptophysin-beads were densely covered by small vesicular profiles with a size distribution typical for synaptic vesicles i.
Rab26 beads were similarly populated with these vesicles, albeit to a lesser extent Figure 1D. Nevertheless, quantitative assessment of the size distribution of the bead-bound vesicles revealed no distinguishable difference between the vesicles bound to synaptophysin and Rab26 beads, respectively Figure 1E.
A Rab26 co-purifies with synaptic vesicles using conventional fractionation. Synaptic vesicles were purified from rat brain homogenate H by two consecutive differential centrifugation steps, yielding a low-speed pellet P1 and a supernatant S1 , followed by a second centrifugation yielding a pellet P2 containing synaptosomes and mitochondria and a supernatant S2.
P2 was then lysed by osmotic shock, followed by centrifugation to separate large particles including synaptic junctional complexes LP1 and a supernatant from which small membranes enriched in synaptic vesicles are collected by high-speed centrifugation LP2, supernatant LS2 only contains soluble proteins.
LP2 was further fractionated by sucrose density gradient centrifugation followed by chromatography on controlled pore glass beads where larger membrane fragments PK1 were separated from synaptic vesicles SV Huttner et al.
Note that Rab26 copurifies with synaptophysin, displaying a pattern typical of synaptic vesicle proteins. The beads were incubated with a resuspended LP2 fraction and collected see [ Burger et al. Note that Rab26 and synaptophysin cofractionate with the immunobeads irrespective of the antibody employed.
Incubation with synaptophysin beads quantitatively depleted Rab26 from the supernatant whereas depletion of synaptophysin by Rabbeads was less complete. In contrast, only the respective antigen was recovered from the detergent-solubilized samples. Asterisks denote IgG heavy and the light chains of the antibodies used for isolation, respectively.
C and D Electron microscopy showing ultrathin sections of methacrylate beads containing bound organelles that were captured with synaptophysin- C or Rabspecific D antibodies, respectively.
E Size distribution diameter of bead-associated vesicles. Note that both populations exhibit a very similar and homogeneous size distribution, with a peak between 40—45 nm as is characteristic for synaptic vesicles Takamori et al.
Membrane association is achieved following posttranslational modification of Rabs by geranyl-geranylation, a prerequisite for membrane insertion and Rab activation. Conversely, membrane dissociation is regulated by a specific adaptor protein, termed GDP dissociation inhibitor GDI , which sequesters GDP-bound Rabs from membranes to the cytosol following GTP-hydrolysis Araki et al.
For this, we incubated a fraction enriched in synaptic vesicles LP2 with purified recombinant GDI in the presence of GDP or GTPγS. Consistent with previous observations, Rab3 is rapidly retrieved from synaptic vesicles membranes by GDI in the presence of GDP Araki et al.
By comparison, Rab26 is resistant to GDI-mediated membrane extraction, even when GDP is present in excess Figure 2A. This feature is reminiscent of the biochemical characteristics of Rab27b, which also fails to be retrieved from synaptic vesicles by GDI treatment in vitro Pavlos et al.
Rather, Rab27b is known to dimerize and persist on synaptic vesicle membranes in its GDP-bound form Chavas et al. As shown in Figure 2B , co-precipitation of FLAG-tagged Rab26 was only observed when cells were transfected with either wild-type EGFP-Rab26 or with a GDP-preferring variant Rab26T77N, henceforth referred to as Rab26TN.
Together, these data indicate that Rab26 is a synaptic vesicle protein that oligomerizes preferentially in its GDP-bound form, thereby precluding GDI-mediated membrane extraction—a feature shared with its synaptic vesicle relative Rab27b.
A Rab26 is resistant to extraction by GDI from synaptic vesicle membranes. An enriched synaptic vesicle fraction LP2 was incubated with GTPγS or GDP µM for 15 min at 37°C. His-GDI 5 µM or PBS control, first lane was added and the samples were incubated for an additional 45 min at 37°C.
The membranes were then separated from the soluble fraction by centrifugation and analyzed by immunoblotting. While Rab3a was efficiently depleted from synaptic vesicles, Rab26 persisted on membranes. IB, immunoblotting. B Rab26 oligomerizes in a GDP-dependent manner. HEK cells transiently co-expressing EGFP-Rab26 variants WT, QL, TN or NI with FLAG-Rab26WT were lysed in detergent containing buffer followed by immunoprecipitation of EGFP-Rabs.
Co-precipitation of FLAG-Rab26WT was observed with EGFP-Rab26 WT and even more efficiently with the GDP-preferring variant Rab26TN whereas co-precipitation with the nucleotide-empty variant Rab26NI was reduced and binding to the GTP-preferring variant Rab26QL was abolished.
IP, immunoprecipitation. Next, to study its subcellular localization in more detail, we immunostained primary cultures of rat hippocampal neurons for Rab First, the distribution of endogenous Rab26 was compared with that of synaptotagmin-I, one of the major membrane constituents of synaptic vesicles.
As shown in Figure 3A , Rab26 labeling resulted in a conspicuous punctate staining pattern that overlapped with, although was not identical to, the pattern obtained with synaptotagmin-I antibodies. Higher magnification of neurites revealed that most of the Rab26 positive puncta colocalized with synaptotagmin-I.
In contrast, many puncta positive for synaptotagmin-I were not stained with the Rab26 antibody Figure 3B , arrows show colocalization. In A — G , representative line scans of the two channels are shown below each set.
In the y-axis, F a. indicates fluorescence intensity arbitrary units. A and B Localization of endogenous Rab26 detected with the newly generated monoclonal anti Rab26 antibody and synaptotagmin-I Syt-I in neurites of dissociated hippocampal neurons DIV 15 reveals that Rab26 colocalizes with a subset of Syt-I positive puncta B , arrows.
C — E Expression of FLAG-tagged Rab26 variants in neurites DIV 9 cultures 48hr after transfection. Both FLAG-Rab26WT C and QL D co-localize with a subset of synaptotagmin positive puncta Syt-I , whereas FLAG-Rab26TN E exhibited a more diffuse distribution.
F Overexpression of EGFP-Rab26WT exhibits a distribution comparable to endogenous and FLAG-tagged Rab26 in neuritis where it colocalizes with synaptophysin Syph. G Rabpositive clusters contain actively recycling vesicles. Hippocampal cultures were incubated overnight with a labeled antibody directed against a lumenal epitope of synaptotagmin Syt-I clone EGFP-Rab26WT is present in recycled synaptic vesicles as evident from the co-localization with synaptotagmin-I positive puncta.
H Localization of YFP-tagged Rab26 variants at the Drosophila neuromuscular junction third instar larvae. The Rab26 variants Rab26wt, GTP-preferring Rab26QL, and GDP-preferring Rab26TN , were expressed using elav-Gal4. Neuromuscular junctions of third instar larvae expressing these Rab26 variants were stained with anti-GFP green and for endogenous cysteine string protein as vesicle marker anti-Csp, red.
To shed more light on the intracellular distribution of Rab26, neurons were transiently transfected with variants of FLAG-tagged Rab26 and then labeled for FLAG and synaptotagmin 1 Figure 3.
The staining pattern obtained with Rab26WT Figure 3C and with Rab26QL the GTP-preferring variant Figure 3D was very similar to that of endogenous Rab26, showing a high degree of overlap with endogenous synaptotagmin 1.
In contrast, the GDP-preferring Rab26TN exhibited a more diffuse but still somewhat granular pattern that displayed no significant co-localization with synaptotagmin 1 Figure 3E.
This suggests that Rab26 is likely to traffic in a pathway distinct from Rab5. Next, we tested whether the Rab26 positive puncta represent synapses undergoing exo-endocytosis.
To monitor this, live hippocampal neuronal cultures expressing EGFP-Rab26 were pre-incubated overnight with a fluorescently-conjugated antibody specific for the luminal domain of synaptotagmin-I. This antibody can only bind to synaptotagmin when the luminal domain of the protein is exposed to the extracellular surface following synaptic vesicle exocytosis, and is thus used to conveniently identify active synapses as well as synaptic vesicles that have undergone exo-endocytosis Kraszewski et al.
As shown in Figure 3G , many of the EGFP-Rab26 puncta colocalized with vesicles labeled with this antibody, thereby confirming that these Rabpositive vesicles originated from endocytosis of vesicles that previously had undergone at least one round of exocytosis.
Rab26 is one of the Rab GTPases conserved between mammals and Drosophila , with the genome of latter encoding three alternatively spliced isoforms. According to a systematic analysis of all Drosophila Rab proteins, Rab26 is expressed specifically in neurons at all developmental stages larval and pupal development, adults flies Chan et al.
To test whether the distribution of Rab26 in Drosophila resembles that in cultured hippocampal neurons, we expressed YFP-tagged versions of wild-type WT , GTP-preferring RabQL and GDP-preferring Rab26TN Rab26 using the pan neuronal elav -Gal4 driver Zhang et al.
Although the tagged Rab26 protein variants were expressed throughout development, no lethality or delayed development was observed data not shown. Consistent with previous observations, analysis of third instar larvae nerve-muscle preparations revealed an exclusive localization of Rab26 to presynaptic compartments of the neuromuscular junction without staining of axons and cell bodies Figure 3—figure supplement 2 ; see also [ Chan et al.
Remarkably, expression of wild-type and of the gain-of-function QL Rab26 resulted in the appearance of large vesicle clusters within the presynaptic boutons whereas the GDP-preferring form TN was diffusely localized.
This indicated that, like in hippocampal neurons, formation of these clusters is dependent on the nucleotide-bound state of Rab26 Figure 3H. These Rabpositive compartments are present in neuromuscular junction boutons as indicated by staining with anti-horseradish peroxidase HRP Figure 3—figure supplement 2 and showed a partial overlap with the synaptic vesicle protein cysteine string protein Csp Figure 3H Zinsmaier et al.
Conspicuously, large Rabpositive structures often showed intense Csp staining at their borders. Importantly, Rab26 is excluded from active zones immunostained for the Bruchpilot Brp , a scaffold protein specifically localized to active zones Figure 3—figure supplement 2 Wagh et al. Whereas the distribution of endogenous and tagged-expressing Rab26 variants was comparable in neurites, expression of EGFP-Rab26WT in cultured hippocampal neurons resulted in a unique and highly conspicuous phenotype that was not observed with endogenous Rab26, untagged or FLAG-tagged Rab26 and that has not been previously reported for any other EGFP-tagged Rab GTPase.
In the soma, EGFP-Rab26WT induced the formation of large vesicular structures that, in some instances, filled the major part of the neuronal cytoplasm Figure 4.
These structures were intensely positive for both synaptic vesicle Figure 4A,B and large dense core vesicle markers, that is, the two types of neuronal secretory vesicles that usually do not show significant overlap Figure 4C and Figure 4—figure supplement 1C.
Intriguingly, the overlap with Rab3A, the major Rab-GTPase associated with synaptic vesicles, was less apparent. Double labeling with a variety of compartment-specific markers revealed no overlap with early endosomes or Golgi Figure 4—figure supplement 1A,B , respectively but some, albeit limited, overlap with lysosomes Figure 4D.
A — C : GFP-positive clusters colocalize with markers for synaptic and large dense core vesicles. A and B EGFP-Rab26WT clusters contain synaptobrevin Sybv and Rab3a.
C Co-expression of EGFP-Rab26WT with RFP-NPY results in almost complete overlap of both proteins in these clusters C. D Partial overlap is also observable with the lysosomal membrane protein LAMP2. DIV 10, scale bar, 5 µm. Line scans of select vesicle clusters denoted by solid white lines in merged panel signify the relative correlation between the individual fluorescent channels.
To better understand the nature of these structures we carried out immunogold-TEM on ultrathin cryosections of transfected neurons. As shown in Figure 5A , the soma contained large clusters of numerous small vesicles that were positive for both EGFP-Rab26WT and synaptobrevin Sybv.
In many cases, these clusters were rather homogenous, but occasionally also contained larger vesicles and mitochondria Figure 5B. Although no systematic quantification was performed, some of these clusters reached enormous dimensions containing possibly s of small vesicles Figure 5—figure supplement 1.
We also assessed neuromuscular junctions of Drosophila strains overexpressing YFP-Rab26WT by TEM. Here, dense clusters of vesicles devoid of surrounding membranes were regularly observable that were clearly set apart from surrounding synaptic vesicles Figure 5D , indicated by an arrow but were clearly absent in controls.
A — C Ultrathin cryosections obtained from hippocampal neurons expressing EGFP-Rab26WT were immunogold labeled for EGFP and synaptobrevin Sybv, monoclonal antibody In the soma of hippocampal neurons organelles surrounded by one or two C , inset, arrow membranes were densely packed with small vesicles and very occasionally other organelles e.
Immunogold labeling for both EGFP and synaptobrevin was concentrated both on vesicles present inside and the surrounding membrane.
Scale bar in insert, 50 nm. D Ultrathin sections of neuromuscular junctions obtained from Drosophila third instar larvae. Control animals show scattered vesicles of a somewhat heterogeneous size, typical for this developmental stage Rasse et al.
Synapses of a strain expressing YFP-Rab26WT using the elavG::UAS system elavG::UAS Rab26WT display frequent clusters of densely packed vesicles arrow that are separated from the surrounding vesicles but lack a surrounding membrane.
As described above, in neurites Rab26 is associated with large clusters containing synaptic vesicle proteins regardless of whether endogenous Rab26 is visualized or whether Rab26 is overexpressed. Thus, it is conceivable that the induction of these vesicle clusters is an intrinsic property of GTP-Rab26, which is enhanced by the weak homo-dimerization of EGFP and YFP Shaner et al.
What could be the identity of these clusters? Autophagy is a degradative pathway during which cellular contents are enclosed by a double-membrane i.
The pathway is initiated by two ubiquitin-like conjugation systems that operate in a sequential manner. The first conjugates the ubiqutin-like protein Atg12 to Atg5 which is then recruited by Atg16L1 to the pre-autophagosome structure.
This complex then recruits a LC3 family member, a second ubiquitin-like molecule, and attaches it covalently to phosphatidylethanolamine in an E3-ligase like reaction Klionsky and Schulman, Since LC3 remains associated with the autophagosomal membrane until its delivery to the lysosome, it is considered to be the most reliable marker for autophagosomes Klionsky et al.
Therefore, to test whether the Rab26 containing clusters are linked to the autophagy pathway, we next checked for association with autophagosome-related proteins.
First, we assessed for colocalization between Rab26 and Atg16L1, a component of pre-autophagosomes Mizushima et al. For this purpose, hippocampal neurons transiently expressing EGFP-Rab26WT were immunostained for endogenous Atg16L1.
Indeed, an almost perfect colocalization between Atg16L1 and Rabpositive clusters was detected in neuronal cell bodies Figure 6A , thereby identifying these clusters as autophagosomal precursors. Next, we stained untransfected neurons for endogenous Rab26 and Atg16L1.
Again, a high degree of overlap was observed between Rab26 and Atg16L1 in clusters decorating neurites Figure 6B but not in cell bodies which remained largely unstained not shown. This indicated that the association of Rab26 with autophagosomes depends on the GTP-form of the protein.
This GTP-dependency was similarly noted in HeLa cells following ectopic expression of Rab In this instance, overexpression of GTP-bound forms WT and QL , but not GDP-bound TN form, of EGFP-Rab26 led to the formation of large Atg16L1-positive clusters Figure 6—figure supplement 1A—C.
Analysis by immunogold-TEM again revealed that these clusters consisted of small but often heterogeneous vesicles, partially surrounded by membranes, with EGFP labeling detected both on vesicles within clusters as well as on their encapsulating membrane s Figure 6—figure supplement 1D.
Arrows indicate co-localization. A Somatic clusters induced by expression of EGFP-Rab26WT co-localize with endogenous Atg16L1. B and C In neurites, Atg16L1 co-localizes with clusters of endogenous Rab26 non-transfected, DIV 15, panel B and with clusters containing FLAG-tagged Rab26WT and Rab26QL, but not with Rab26TN transfected, DIV 9, panel C.
D Similar colocalization patterns were obtained from neurites expressing FLAG-Rab26 variants and autophagosomes labeled by GFP-LC3B. Note that occasional puncta were observed for the GDP-preferring variant Rab26TN that, however, showed no overlap with LC3B arrowhead.
DIV 9. E and F Co-expression of FLAG-Rab26WT and EGFP-Rab33WT in hippocampal neurons. In the soma E , EGFP-Rab33 in primary restricted to a perinuclear structure reminiscent of the Golgi apparatus whereas an almost perfect overlap was observed between the Rab33 and Rab26 in peripheral puncta lining neurites F.
DIV 8. Transient association of Atg16L1 to pre-autophagosomal structures enables the recruitment and membrane attachment of LC3 family members that persist on the autophagosomal membranes until degradation. Co-expression of GFP-LC3 and active forms FLAG-Rab26 WT and QL resulted in a localization pattern comparable to that observed for Atg16L1 Figure 6D , thereby verifying the nature of these compartments as autophagosomes.
Indeed, there is now a growing body evidence implicating several Rabs Rab1, Rab7, Rab9, Rab11, Rab24, Rab32, and Rab33, inclusive in canonical autophagy for a comprehensive review [ Chua et al.
Among these, Rab33, a Golgi resident Rab, participates in the formation of autophagosome precursors by recruiting Atg16L1 a Rab33 effector to isolation membranes Itoh et al.
Small Autophayy of the Rab family Tips for proper rehydration only Autohagy target recognition in membrane Autophagt but also control other cellular functions such Autophagy and GTPases cytoskeletal Quercetin and energy boost and autophagy. Here we show Tips for proper rehydration Rab26 is specifically associated with Autophagh of synaptic vesicles in neurites. Overexpression of active but not of GDP-preferring Rab26 enhances vesicle clustering, which is particularly conspicuous for the EGFP-tagged variant, resulting in a massive accumulation of synaptic vesicles in neuronal somata without altering the distribution of other organelles. Both endogenous and induced clusters co-localize with autophagy-related proteins such as Atg16L1, LC3B and Rab33B but not with other organelles. Furthermore, Atg16L1 appears to be a direct effector of Rab26 and binds Rab26 in its GTP-bound form, albeit only with low affinity.Autophagy and GTPases -
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Abstract Autophagy is a conserved cellular degradation process in eukaryotes that facilitates the recycling and reutilization of damaged organelles and compartments. Background Autophagy and its machinery Autophagy is a cellular degradative pathway involving the delivery of cytoplasmic cargo to lysosomes and is highly conserved among eukaryotes ranging from yeast to mammalian cells [ 1 , 2 , 3 ].
Role of Rab proteins in vesicle transport Rab proteins comprise a large family of small guanosine triphosphate GTP -binding proteins that play a crucial role in the regulation of intracellular vesicle trafficking [ 31 , 32 , 33 ]. Full size image.
Main text Rab1 Rab1 GTPase 22 kDa localized in the ER-Golgi intermediate is involved in the regulation of membrane trafficking from the ER to the Golgi [ 45 , 46 ].
Rab6 Rab6 GTPase 24 kDa with its intra-Golgi localization, serves as a trans-Golgi marker with a well-established role in retrograde transport within the Golgi and between the Golgi and ER or endosome membrane [ 58 , 59 , 60 ].
Rab9 Rab9 GTPase 23 kDa localized in the late endosome plays an important role in vesicle transport from the late endosome to the trans-Golgi network TGN [ 68 ].
Rab30 Rab30 GTPase 23 kDa localized in trans-Golgi and is commonly found in metazoans. Rab33 Rab33 GTPase 27 kDa localized in the Golgi apparatus is involved in intra-Golgi transport. Rab37 Rab37 GTPase 25 kDa localized in the Golgi apparatus participates in autophagosome formation [ ].
Conclusion and future perspectives The involvement of Golgi-associated Rab GTPases in the initiation, formation, maturation, and fusion of autophagosome summarized in this review suggests the crucial role played by them in autophagy.
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Less is known, however, about the molecular cascades involved in synaptic elimination. Elimination from the outside is usually executed by microglial cells but the underlying signaling network is complex, and astrocytes have also recently been appreciated to play a major role in this process Chung and Barres, ; Stephan et al.
In cell autonomous elimination, synaptic components may either be i recycled, that is, being removed in a functionally intact form for the use at another site, or ii degraded.
With the exception of some recent evidence showing that synaptic vesicles can be exchanged between neighboring synapses, whether synaptic components can be reused after having been operational in a functional synapse remains largely unexplored Darcy et al.
Conversely, an increasing body of evidence supports the view that synaptic components are rapidly degraded once a synapse is earmarked for elimination.
Unsurprisingly, the ubiquitin-proteasome system is emerging as one of the central players, both at presynaptic Yao et al. At the presynaptic site, the ubiquitin system is not only involved in synaptic elimination, but also in the general regulation of synaptic plasticity Muralidhar and Thomas, ; Campbell and Holt, ; DiAntonio et al.
For instance the protein RIM, a crucial hub for organizing active zones that form the release site for synaptic vesicles, was recently shown to be rapidly turned over upon ubiquitination, resulting in loss of synaptic function Yao et al. Furthermore, an increasing number of ubiquitin-modifying enzymes have been described from synapses particularly E3-ligases Ding and Shen, In contrast to the emerging role of the ubiquitin proteasome system, little information is currently available regarding the mechanisms by which synaptic membrane proteins are eliminated.
At the postsynaptic site, ubiquitin-dependent pathways are clearly involved in the regulation of surface receptor density Patrick et al. However, only scant information is available about the turnover of membrane proteins at the presynaptic site where a complicated and autonomous vesicle recycling machinery needs to deal with many s of synaptic vesicles.
Surprisingly, the mechanisms by which synaptic vesicles are eliminated have thus far received little attention. By analogy to non-neuronal cells, it is frequently assumed that synaptic vesicle membrane proteins follow the canonical endosomal-lysosomal route for degradation which involves ubiquitination and recognition by the ESCRT machinery after being delivered to endosomes, followed by the formation of multivesicular bodies, retrograde transport, and ultimately fusion with lysosomes Katzmann et al.
However, aside from a few hints from a recent proteomic study, whether synaptic vesicle proteins are ubiquitinated remains unclear Na et al.
Similarly, whether sequestration into the lumen of multivesicular bodies is involved and, if so, to what extent is unknown. Indeed, multivesicular bodies are infrequently observed in axons and typically appear in response to pathological dystrophic or toxic conditions for review see [ Von Bartheld and Altick, ].
Furthermore, no information is currently available concerning the involvement of the ESCRT pathway in the elimination of presynaptic components.
Intriguingly, recent studies implicate the involvement of clathrin-dependent pathways in targeting plasma membrane components to autophagosomes, hinting at the potential involvement of this mechanism in the turnover of synaptic vesicles recovered by endocytosis following the release of their neurotransmitter content Ravikumar et al.
In this study we report about data suggesting the presence of a novel pathway for the degradation of synaptic and secretory vesicles, which involves selective sequestration of vesicle clusters into structures resembling early autophagosomes.
We show that Rab26 selectively localizes to presynaptic membrane vesicles and recruits both Atg16L1 and Rab33B, two components of the pre-autophagosomal machinery. Remarkably, these autophagosomal structures are filled almost exclusively with synaptic vesicles and proteins typically associated with large dense-core vesicles.
Overexpression of EGFP-tagged Rab26, but not of FLAG-tagged or wild-type WT Rab26, induces the formation of giant autophagosomes in the cell bodies of hippocampal neurons—a phenotype that is mirrored upon transfection in HeLa cells.
Based on these findings, we conclude that Rab26 may selectively channel synaptic vesicles into pre-autophagosomes and, thus, may represent a new regulator of synapse turnover. Previously we reported that synaptic vesicles highly purified from rat brain contain more than 30 different Rab-GTPases Takamori et al.
Of these, a subgroup of Rabs including Rab3a, Rab3b, Rab3c, and Rab27b were highly enriched in the vesicle fraction Pavlos et al. Rab26, a comparatively uncharacterized member of the Rab superfamily, is also closely related to this subgroup.
Since we detected Rab26 on purified synaptic vesicles in two previous independent proteomic studies Takamori et al. To this end, we raised a mouse monoclonal antibody that is specific for Rab26 and does not cross-react with other related Rab proteins including Rab27 Figure 1—figure supplement 1.
First, we used immunoblotting to monitor the subcellular distribution of Rab26 during the purification of synaptic vesicles from the rat brain.
As shown in Figure 1A , Rab26 co-purified with synaptic vesicle markers as indicated here using synaptophysin , with the highest enrichment being observed in the synaptic vesicle SV fraction obtained after purification using consecutive density gradients and size exclusion chromatography.
For independent confirmation, we carried out immunoisolation of synaptic vesicles using beads Eupergit C1Z covalently coupled with monoclonal antibodies specific for Rab26 or synaptophysin. As shown in Figure 1B , both antibodies resulted in the isolation of membranes highly enriched in both synaptophysin and Rab As a control, the membranes were solubilized with the detergent Triton X prior to immunoisolation Tx-IP.
In this case, only the respective antigens were isolated Figure 1B , thus validating the specificity of the isolation procedure.
We also verified the nature of the immunoisolated vesicles by transmission electron microscopy TEM. As previously reported, synaptophysin-beads were densely covered by small vesicular profiles with a size distribution typical for synaptic vesicles i.
Rab26 beads were similarly populated with these vesicles, albeit to a lesser extent Figure 1D. Nevertheless, quantitative assessment of the size distribution of the bead-bound vesicles revealed no distinguishable difference between the vesicles bound to synaptophysin and Rab26 beads, respectively Figure 1E.
A Rab26 co-purifies with synaptic vesicles using conventional fractionation. Synaptic vesicles were purified from rat brain homogenate H by two consecutive differential centrifugation steps, yielding a low-speed pellet P1 and a supernatant S1 , followed by a second centrifugation yielding a pellet P2 containing synaptosomes and mitochondria and a supernatant S2.
P2 was then lysed by osmotic shock, followed by centrifugation to separate large particles including synaptic junctional complexes LP1 and a supernatant from which small membranes enriched in synaptic vesicles are collected by high-speed centrifugation LP2, supernatant LS2 only contains soluble proteins.
LP2 was further fractionated by sucrose density gradient centrifugation followed by chromatography on controlled pore glass beads where larger membrane fragments PK1 were separated from synaptic vesicles SV Huttner et al. Note that Rab26 copurifies with synaptophysin, displaying a pattern typical of synaptic vesicle proteins.
The beads were incubated with a resuspended LP2 fraction and collected see [ Burger et al. Note that Rab26 and synaptophysin cofractionate with the immunobeads irrespective of the antibody employed.
Incubation with synaptophysin beads quantitatively depleted Rab26 from the supernatant whereas depletion of synaptophysin by Rabbeads was less complete. In contrast, only the respective antigen was recovered from the detergent-solubilized samples. Asterisks denote IgG heavy and the light chains of the antibodies used for isolation, respectively.
C and D Electron microscopy showing ultrathin sections of methacrylate beads containing bound organelles that were captured with synaptophysin- C or Rabspecific D antibodies, respectively. E Size distribution diameter of bead-associated vesicles. Note that both populations exhibit a very similar and homogeneous size distribution, with a peak between 40—45 nm as is characteristic for synaptic vesicles Takamori et al.
Membrane association is achieved following posttranslational modification of Rabs by geranyl-geranylation, a prerequisite for membrane insertion and Rab activation. Conversely, membrane dissociation is regulated by a specific adaptor protein, termed GDP dissociation inhibitor GDI , which sequesters GDP-bound Rabs from membranes to the cytosol following GTP-hydrolysis Araki et al.
For this, we incubated a fraction enriched in synaptic vesicles LP2 with purified recombinant GDI in the presence of GDP or GTPγS.
Consistent with previous observations, Rab3 is rapidly retrieved from synaptic vesicles membranes by GDI in the presence of GDP Araki et al.
By comparison, Rab26 is resistant to GDI-mediated membrane extraction, even when GDP is present in excess Figure 2A. This feature is reminiscent of the biochemical characteristics of Rab27b, which also fails to be retrieved from synaptic vesicles by GDI treatment in vitro Pavlos et al.
Rather, Rab27b is known to dimerize and persist on synaptic vesicle membranes in its GDP-bound form Chavas et al. As shown in Figure 2B , co-precipitation of FLAG-tagged Rab26 was only observed when cells were transfected with either wild-type EGFP-Rab26 or with a GDP-preferring variant Rab26T77N, henceforth referred to as Rab26TN.
Together, these data indicate that Rab26 is a synaptic vesicle protein that oligomerizes preferentially in its GDP-bound form, thereby precluding GDI-mediated membrane extraction—a feature shared with its synaptic vesicle relative Rab27b. A Rab26 is resistant to extraction by GDI from synaptic vesicle membranes.
An enriched synaptic vesicle fraction LP2 was incubated with GTPγS or GDP µM for 15 min at 37°C. His-GDI 5 µM or PBS control, first lane was added and the samples were incubated for an additional 45 min at 37°C. The membranes were then separated from the soluble fraction by centrifugation and analyzed by immunoblotting.
While Rab3a was efficiently depleted from synaptic vesicles, Rab26 persisted on membranes. IB, immunoblotting. B Rab26 oligomerizes in a GDP-dependent manner. HEK cells transiently co-expressing EGFP-Rab26 variants WT, QL, TN or NI with FLAG-Rab26WT were lysed in detergent containing buffer followed by immunoprecipitation of EGFP-Rabs.
Co-precipitation of FLAG-Rab26WT was observed with EGFP-Rab26 WT and even more efficiently with the GDP-preferring variant Rab26TN whereas co-precipitation with the nucleotide-empty variant Rab26NI was reduced and binding to the GTP-preferring variant Rab26QL was abolished.
IP, immunoprecipitation. Next, to study its subcellular localization in more detail, we immunostained primary cultures of rat hippocampal neurons for Rab First, the distribution of endogenous Rab26 was compared with that of synaptotagmin-I, one of the major membrane constituents of synaptic vesicles.
As shown in Figure 3A , Rab26 labeling resulted in a conspicuous punctate staining pattern that overlapped with, although was not identical to, the pattern obtained with synaptotagmin-I antibodies. Higher magnification of neurites revealed that most of the Rab26 positive puncta colocalized with synaptotagmin-I.
In contrast, many puncta positive for synaptotagmin-I were not stained with the Rab26 antibody Figure 3B , arrows show colocalization. In A — G , representative line scans of the two channels are shown below each set.
In the y-axis, F a. indicates fluorescence intensity arbitrary units. A and B Localization of endogenous Rab26 detected with the newly generated monoclonal anti Rab26 antibody and synaptotagmin-I Syt-I in neurites of dissociated hippocampal neurons DIV 15 reveals that Rab26 colocalizes with a subset of Syt-I positive puncta B , arrows.
C — E Expression of FLAG-tagged Rab26 variants in neurites DIV 9 cultures 48hr after transfection. Both FLAG-Rab26WT C and QL D co-localize with a subset of synaptotagmin positive puncta Syt-I , whereas FLAG-Rab26TN E exhibited a more diffuse distribution. F Overexpression of EGFP-Rab26WT exhibits a distribution comparable to endogenous and FLAG-tagged Rab26 in neuritis where it colocalizes with synaptophysin Syph.
G Rabpositive clusters contain actively recycling vesicles. Hippocampal cultures were incubated overnight with a labeled antibody directed against a lumenal epitope of synaptotagmin Syt-I clone EGFP-Rab26WT is present in recycled synaptic vesicles as evident from the co-localization with synaptotagmin-I positive puncta.
H Localization of YFP-tagged Rab26 variants at the Drosophila neuromuscular junction third instar larvae. The Rab26 variants Rab26wt, GTP-preferring Rab26QL, and GDP-preferring Rab26TN , were expressed using elav-Gal4.
Neuromuscular junctions of third instar larvae expressing these Rab26 variants were stained with anti-GFP green and for endogenous cysteine string protein as vesicle marker anti-Csp, red.
To shed more light on the intracellular distribution of Rab26, neurons were transiently transfected with variants of FLAG-tagged Rab26 and then labeled for FLAG and synaptotagmin 1 Figure 3.
The staining pattern obtained with Rab26WT Figure 3C and with Rab26QL the GTP-preferring variant Figure 3D was very similar to that of endogenous Rab26, showing a high degree of overlap with endogenous synaptotagmin 1.
In contrast, the GDP-preferring Rab26TN exhibited a more diffuse but still somewhat granular pattern that displayed no significant co-localization with synaptotagmin 1 Figure 3E. This suggests that Rab26 is likely to traffic in a pathway distinct from Rab5. Next, we tested whether the Rab26 positive puncta represent synapses undergoing exo-endocytosis.
To monitor this, live hippocampal neuronal cultures expressing EGFP-Rab26 were pre-incubated overnight with a fluorescently-conjugated antibody specific for the luminal domain of synaptotagmin-I.
This antibody can only bind to synaptotagmin when the luminal domain of the protein is exposed to the extracellular surface following synaptic vesicle exocytosis, and is thus used to conveniently identify active synapses as well as synaptic vesicles that have undergone exo-endocytosis Kraszewski et al.
As shown in Figure 3G , many of the EGFP-Rab26 puncta colocalized with vesicles labeled with this antibody, thereby confirming that these Rabpositive vesicles originated from endocytosis of vesicles that previously had undergone at least one round of exocytosis.
Rab26 is one of the Rab GTPases conserved between mammals and Drosophila , with the genome of latter encoding three alternatively spliced isoforms. According to a systematic analysis of all Drosophila Rab proteins, Rab26 is expressed specifically in neurons at all developmental stages larval and pupal development, adults flies Chan et al.
To test whether the distribution of Rab26 in Drosophila resembles that in cultured hippocampal neurons, we expressed YFP-tagged versions of wild-type WT , GTP-preferring RabQL and GDP-preferring Rab26TN Rab26 using the pan neuronal elav -Gal4 driver Zhang et al.
Although the tagged Rab26 protein variants were expressed throughout development, no lethality or delayed development was observed data not shown. Consistent with previous observations, analysis of third instar larvae nerve-muscle preparations revealed an exclusive localization of Rab26 to presynaptic compartments of the neuromuscular junction without staining of axons and cell bodies Figure 3—figure supplement 2 ; see also [ Chan et al.
Remarkably, expression of wild-type and of the gain-of-function QL Rab26 resulted in the appearance of large vesicle clusters within the presynaptic boutons whereas the GDP-preferring form TN was diffusely localized. This indicated that, like in hippocampal neurons, formation of these clusters is dependent on the nucleotide-bound state of Rab26 Figure 3H.
These Rabpositive compartments are present in neuromuscular junction boutons as indicated by staining with anti-horseradish peroxidase HRP Figure 3—figure supplement 2 and showed a partial overlap with the synaptic vesicle protein cysteine string protein Csp Figure 3H Zinsmaier et al.
Conspicuously, large Rabpositive structures often showed intense Csp staining at their borders. Importantly, Rab26 is excluded from active zones immunostained for the Bruchpilot Brp , a scaffold protein specifically localized to active zones Figure 3—figure supplement 2 Wagh et al.
Whereas the distribution of endogenous and tagged-expressing Rab26 variants was comparable in neurites, expression of EGFP-Rab26WT in cultured hippocampal neurons resulted in a unique and highly conspicuous phenotype that was not observed with endogenous Rab26, untagged or FLAG-tagged Rab26 and that has not been previously reported for any other EGFP-tagged Rab GTPase.
In the soma, EGFP-Rab26WT induced the formation of large vesicular structures that, in some instances, filled the major part of the neuronal cytoplasm Figure 4. These structures were intensely positive for both synaptic vesicle Figure 4A,B and large dense core vesicle markers, that is, the two types of neuronal secretory vesicles that usually do not show significant overlap Figure 4C and Figure 4—figure supplement 1C.
Intriguingly, the overlap with Rab3A, the major Rab-GTPase associated with synaptic vesicles, was less apparent. Double labeling with a variety of compartment-specific markers revealed no overlap with early endosomes or Golgi Figure 4—figure supplement 1A,B , respectively but some, albeit limited, overlap with lysosomes Figure 4D.
A — C : GFP-positive clusters colocalize with markers for synaptic and large dense core vesicles. A and B EGFP-Rab26WT clusters contain synaptobrevin Sybv and Rab3a.
C Co-expression of EGFP-Rab26WT with RFP-NPY results in almost complete overlap of both proteins in these clusters C. D Partial overlap is also observable with the lysosomal membrane protein LAMP2.
DIV 10, scale bar, 5 µm. Line scans of select vesicle clusters denoted by solid white lines in merged panel signify the relative correlation between the individual fluorescent channels.
To better understand the nature of these structures we carried out immunogold-TEM on ultrathin cryosections of transfected neurons.
As shown in Figure 5A , the soma contained large clusters of numerous small vesicles that were positive for both EGFP-Rab26WT and synaptobrevin Sybv. In many cases, these clusters were rather homogenous, but occasionally also contained larger vesicles and mitochondria Figure 5B.
Although no systematic quantification was performed, some of these clusters reached enormous dimensions containing possibly s of small vesicles Figure 5—figure supplement 1. We also assessed neuromuscular junctions of Drosophila strains overexpressing YFP-Rab26WT by TEM.
Here, dense clusters of vesicles devoid of surrounding membranes were regularly observable that were clearly set apart from surrounding synaptic vesicles Figure 5D , indicated by an arrow but were clearly absent in controls.
A — C Ultrathin cryosections obtained from hippocampal neurons expressing EGFP-Rab26WT were immunogold labeled for EGFP and synaptobrevin Sybv, monoclonal antibody In the soma of hippocampal neurons organelles surrounded by one or two C , inset, arrow membranes were densely packed with small vesicles and very occasionally other organelles e.
Immunogold labeling for both EGFP and synaptobrevin was concentrated both on vesicles present inside and the surrounding membrane. Scale bar in insert, 50 nm. D Ultrathin sections of neuromuscular junctions obtained from Drosophila third instar larvae.
Control animals show scattered vesicles of a somewhat heterogeneous size, typical for this developmental stage Rasse et al. Synapses of a strain expressing YFP-Rab26WT using the elavG::UAS system elavG::UAS Rab26WT display frequent clusters of densely packed vesicles arrow that are separated from the surrounding vesicles but lack a surrounding membrane.
As described above, in neurites Rab26 is associated with large clusters containing synaptic vesicle proteins regardless of whether endogenous Rab26 is visualized or whether Rab26 is overexpressed. Thus, it is conceivable that the induction of these vesicle clusters is an intrinsic property of GTP-Rab26, which is enhanced by the weak homo-dimerization of EGFP and YFP Shaner et al.
What could be the identity of these clusters? Autophagy is a degradative pathway during which cellular contents are enclosed by a double-membrane i. The pathway is initiated by two ubiquitin-like conjugation systems that operate in a sequential manner.
The first conjugates the ubiqutin-like protein Atg12 to Atg5 which is then recruited by Atg16L1 to the pre-autophagosome structure. This complex then recruits a LC3 family member, a second ubiquitin-like molecule, and attaches it covalently to phosphatidylethanolamine in an E3-ligase like reaction Klionsky and Schulman, Since LC3 remains associated with the autophagosomal membrane until its delivery to the lysosome, it is considered to be the most reliable marker for autophagosomes Klionsky et al.
Therefore, to test whether the Rab26 containing clusters are linked to the autophagy pathway, we next checked for association with autophagosome-related proteins. First, we assessed for colocalization between Rab26 and Atg16L1, a component of pre-autophagosomes Mizushima et al.
For this purpose, hippocampal neurons transiently expressing EGFP-Rab26WT were immunostained for endogenous Atg16L1. Indeed, an almost perfect colocalization between Atg16L1 and Rabpositive clusters was detected in neuronal cell bodies Figure 6A , thereby identifying these clusters as autophagosomal precursors.
Next, we stained untransfected neurons for endogenous Rab26 and Atg16L1. Again, a high degree of overlap was observed between Rab26 and Atg16L1 in clusters decorating neurites Figure 6B but not in cell bodies which remained largely unstained not shown.
This indicated that the association of Rab26 with autophagosomes depends on the GTP-form of the protein. This GTP-dependency was similarly noted in HeLa cells following ectopic expression of Rab In this instance, overexpression of GTP-bound forms WT and QL , but not GDP-bound TN form, of EGFP-Rab26 led to the formation of large Atg16L1-positive clusters Figure 6—figure supplement 1A—C.
Analysis by immunogold-TEM again revealed that these clusters consisted of small but often heterogeneous vesicles, partially surrounded by membranes, with EGFP labeling detected both on vesicles within clusters as well as on their encapsulating membrane s Figure 6—figure supplement 1D.
Arrows indicate co-localization. A Somatic clusters induced by expression of EGFP-Rab26WT co-localize with endogenous Atg16L1. B and C In neurites, Atg16L1 co-localizes with clusters of endogenous Rab26 non-transfected, DIV 15, panel B and with clusters containing FLAG-tagged Rab26WT and Rab26QL, but not with Rab26TN transfected, DIV 9, panel C.
D Similar colocalization patterns were obtained from neurites expressing FLAG-Rab26 variants and autophagosomes labeled by GFP-LC3B. Note that occasional puncta were observed for the GDP-preferring variant Rab26TN that, however, showed no overlap with LC3B arrowhead.
DIV 9. E and F Co-expression of FLAG-Rab26WT and EGFP-Rab33WT in hippocampal neurons. In the soma E , EGFP-Rab33 in primary restricted to a perinuclear structure reminiscent of the Golgi apparatus whereas an almost perfect overlap was observed between the Rab33 and Rab26 in peripheral puncta lining neurites F.
DIV 8. Transient association of Atg16L1 to pre-autophagosomal structures enables the recruitment and membrane attachment of LC3 family members that persist on the autophagosomal membranes until degradation. Co-expression of GFP-LC3 and active forms FLAG-Rab26 WT and QL resulted in a localization pattern comparable to that observed for Atg16L1 Figure 6D , thereby verifying the nature of these compartments as autophagosomes.
Indeed, there is now a growing body evidence implicating several Rabs Rab1, Rab7, Rab9, Rab11, Rab24, Rab32, and Rab33, inclusive in canonical autophagy for a comprehensive review [ Chua et al. Among these, Rab33, a Golgi resident Rab, participates in the formation of autophagosome precursors by recruiting Atg16L1 a Rab33 effector to isolation membranes Itoh et al.
Given that Rab26 colocalizes with Atg16L1, we checked for potential cooperation between Rab26 and Rab33 in neurons. For this, hippocampal neurons were co-transfected with FLAG-Rab26WT and EGFP-Rab33BWT.
On the other hand, significant overlap between Rab26 and Rab33 was observed in more peripheral puncta lining neurites Figure 6F. Together these data imply that the autophagy-pathway regulated by Rab26 may functionally intersect with Rab The overlap between Rab26 and Rab33 prompted us to further investigate whether Atg16L1 may also be an effector of Rab To explore this possibility, we performed co-immunoprepitation experiments between FLAG-tagged Rab26 WT, QL or TN and endogenous Atg16L1 in HeLa cells.
As shown in Figure 7A , all three FLAG-tagged Rab26 variants were efficiently immunoprecipitated with the FLAG antibody. Immunoblotting for endogenous Atg16L1 from the same immunoprecipitates revealed co-precipitation between Atg16L1 and Rab26QL.
By comparison, little to no Atg16L1 was detectable in the precipitates of RabWT and Rab26TN, respectively, indicating that the interaction between Rab26 and Atg16L1 is GTP-dependent. A Co-Immunoprecipitation of FLAG-tagged Rab26 variants expressed in HeLa cells with endogenous Atg16L1 protein.
Immunoprecipitation was carried out following lysis in detergent-containing buffer and clearance by centrifugation to remove cell debris. Note that only the GTP-preferring QL variant of Rab26 showed significant binding to Atg16L1 arrow. B GST pulldown of purified recombinantly expressed GST-Rab26 variants with a pre-formed complex of His-tagged versions of Atg5 and the N-terminal domain of Atg16L1 Atg16NT.
Note that Atg16NT selectively interacted with the GTP-preferring QL-variant of Rab In parallel, we performed GST-pulldown assays to verify the results from the coIP experiments.
For this, purified bacterially expressed recombinant Rab26 variants QL or TN , tagged with GST were incubated with a preassembled complex of Atg5 and the N-terminal fragment of Atg16L1 containing its coiled coil domain Atg16NT. In agreement with our immunoprecipitation studies, GST-pulldown revealed an interaction between Atg16L1 and Rab26, with Atg16L1 binding to the QL and to a lesser extent the TN-variant of Rab26 Figure 7B , with the latter being further reduced upon repetitive washings not shown.
Atg5 remains bound in this complex. To further examine the interaction, we analyzed the binding between Rab26 and Atg16L1 using analytical gel filtration. Surprisingly, formation of Rab26 QL -ATG16L1 complexes were not detectable with this approach Figure 7—figure supplement 1.
As a positive control, we carried out the same experiment using Rab33 QL and ATG16L1. Here, complex formation was detectable with this approach. Thus, while both IP and pull-down experiments show that RAB26 binds ATG16L1 in a GTP-dependent manner, this binding appears to be weaker than the interaction between Rab33 and ATG16L1.
In the present study we have combined multiple complementary biochemical and cell biological approaches to demonstrate that the small GTPase Rab26 is specifically associated with synaptic vesicles. Intriguingly, Rab26 appears to be particularly enriched in large clusters of synaptic vesicles to which the autophagy proteins Atg16L1, LC3 and Rab33B are recruited, suggesting that they represent pre-autophagosomal compartments.
We show further that, at least when using overexpression of EGFP-tagged Rab26, such clusters are also formed in cell bodies where they are enclosed by a single and in some instances a double isolation membrane.
Rab26 is most closely related to the secretory GTPases Rab3 and Rab27, which led to the conclusion that it may perform similar functions in membrane traffic Fukuda, This view is supported by reports showing association of Rab26 with zymogen granules in exocrine cells Nashida et al.
More recently, Rab26 has been found to be associated with lysosomes in zymogen-secreting cells Jin and Mills, implying that its functions in secretory cells extend beyond that of exocytosis.
In our previous work Takamori et al. Our present data now show that this association is exclusive, with Rab26 being absent from other organelles such as early endosomes, paralleling the distribution of other secretory Rabs. On the other hand, the preferential association of Rab26 with large clusters of synaptic vesicles and its conspicuous absence from smaller boutons positive for synaptic vesicle markers is clearly distinct from Rab3 and Rab27b and indicates that Rab26 may not be contributing to the canonical function of these Rabs in regulated exocytosis.
Intriguingly, in contrast to for example, Rab3 and Rab5, Rab26 cannot be extracted from synaptic vesicle membranes by GDI in its GDP-form—a feature it shares with Rab27b.
Rather, Rab26 exhibits a tendency to oligomerize in the GDP-form, again a feature shared with Rab27b and perhaps with some others such as Rab11 and Rab9, which crystallize as dimers in the GDP-state Pasqualato et al. It is somewhat surprising that, along with the GDP-bound variant, wild-type Rab26 also appears to oligomerize albeit to a lesser extent.
Perhaps the most conspicuous feature of Rab26 is that it is not only preferentially associated with secretory vesicle clusters but actually induces their formation in a GTP-dependent manner as becomes apparent upon the expression of exogenous Rab26 variants in both neurons and non-neuronal cells.
This is most dramatically observed with the EGFP-tagged variant suggesting that the weak homodimerization tendency of EGFP enhances the phenotype note that no other EGFP-tagged Rab exhibits similar features including the most abundant secretory GTPase, Rab3a.
At present, the exact mechanism underlying this clustering phenotype is unclear. Nevertheless, since the GTP-form of Rab26 does not oligomerize, it is unlikely that clustering is effected by homophilic Rab26 interactions. Rather, it possible that clustering is mediated by a hitherto unknown effector protein.
This effector is probably distinct from Atg16L1 as overexpression of EGFP-Rab33B that also recruits Atg16L1 does not induce such clusters Figure 6 , and data not shown.
However, given that the central terminal region of Atg16L1 has a tendency for homo-multimerization, this possibility cannot be excluded Mizushima et al. Intriguingly, our findings agree with a recent report according to which overexpression of Rab26 in exocrine cell lines induces clustering of lysosomes, reminiscent of the partial co-localization of the EGFP-induced Rab26 clusters with lysosomes in neuronal cell bodies Jin and Mills, Our results indicate that the core autophagy protein Atg16L1 is an effector of Rab26 that binds to the GTPase exclusively in the GTP-form, paralleling previous findings on the Golgi-resident Rab33B Itoh et al.
Interestingly, binding of Rab26 to Atg16L1 appears to be weaker than that between Rab33 and Atg16L1, which plays a role in canonical autophagy, probably explaining why Itoh et al. It is conceivable that the interaction is more transient, or else, that it requires additional factors for stabilization, thus allowing for fine-tuning the flow of synaptic vesicles targeted for selective autophagy.
How does recruitment of Atg16L1 to synaptic vesicle clusters relate to the established steps of autophagosome formation? First of all, it cannot yet be excluded with certainty that upon recruitment to these vesicles Atg16L1 performs a non-canonical function that is not related to autophagosome formation see e.
In particular, Atg16L1 and Rab33A have recently been found to be associated with secretory vesicles in neuroendocrine PC12 cells, with the data suggesting a role for Atg16L1 in regulating exocytosis independent of autophagy Ishibashi et al.
On the other hand, based on our extensive morphological assessment using double immunolabeling microscopy, we strongly favor that the RabAtg16L1 complexes in neurons represent pre-autophagosomal structures because i Rab26 is not present on all synaptic vesicles but rather confined to vesicle aggregates that may be functionally impaired, and ii LC3 is recruited to these clusters suggesting that the formation of an autophagosomal membrane is, at least in part, initiated.
Our data indicates that the vesicle clusters containing Rab26 and Atg16L1 have undergone exo-endocytotic cycling. Intriguingly, clathrin has recently been shown to interact with Atg16L1, thus targeting plasma membrane constituents towards autophagosome precursors via clathrin-mediated endocytosis Ravikumar et al.
Since clathrin-mediated endocytosis constitutes the main endocytotic pathway for synaptic vesicles, it is conceivable that there is a synergy between Rab and clathrin-induced autophagocytosis in nerve terminals that further fine-tunes the targeting of synaptic vesicles to preautophagosomal structures.
In many of these cases the pathway is initiated by ubiquitination of target proteins. While we do not know whether this is also the case here, it is conceivable that the initiation event may indeed be the recruitment of active Rab26 to the membrane of subsets of synaptic vesicles that then interacts with other factors to form clusters and to recruit an isolation membrane, the origin of which remains to be identified.
Following the classical work in the early 70s of last century demonstrating that synaptic vesicles undergo multiple rounds of recycling in the synapse, Atwood et al. However, all membrane constituents age and accumulate structural defects requiring sorting out of damaged constituents.
Although no increase in the number of late endosomes, lysosomes or autophagosomes was observed following even massive stimulation, it was hypothesized as early as that newly reformed synaptic vesicles could either be actively re-used as functional synaptic vesicles or re-directed to a pathway ultimately leading to lysosomes as the final destination for degradation Holtzman et al.
Our discovery of vesiculophagy as a pathway initiated in presynaptic boutons that directs entire synaptic vesicle pools towards autophagosomes provides a previously uncharacterized link towards lysosomal degradation of trafficking organelles which is distinct from the classical endosomal route.
Indeed, recent data suggest that presynaptic neurotransmission is functionally modulated by macroautophagy. Induction of autophagy in neurons increased the amount of autophagic vacuoles in presynaptic terminals and with an accompanying reduction in synaptic vesicle number and decrease in evoked neurotransmitter release Hernandez et al.
Furthermore, two groups have recently suggested that in axons autophagosomes originate distally and then are transported by retrograde motors towards the cell body.
During their travel they undergo fusion with acidic compartments and finally with the lysosomes Lee et al. It is therefore conceivable that Rab26 feeds vesicle membranes into autophagosomes that may form and mature during retrograde transport.
How this novel pathway is initiated and regulated will be the subject of future studies. Mouse monoclonal and rabbit polyclonal antibodies specific for synaptophysin, synaptotagmin, synaptobrevin, Rab3a, GDI Cl Mouse anti-LAMP2 antibody was purchased from the Developmental Studies Hybridoma Bank DSHB, University of Iowa, IA.
Antibodies against EEA1 and GM were purchased from BD Bioscience Franklin Lakes, NJ. The antibody against the FLAG epitope was obtained from Stratagene La Jolla, CA. Antibodies specific for Atg16L1 were purchased from CosmoBio Tokyo and MBL Nagoya.
Anti-Atg5 antibody was from Novus Biological Littleton, Colorado. The antibody against secretogranin II was kindly provided by Sharon Tooze Cancer Research UK. Cells from knee lymph nodes were fused with the mouse myeloma cell line P3X63Ag.
Cell culture supernatants obtained from individual clones were then screened using enzyme-linked immunosorbent assay ELISA , immunoblot assays and immunoflourescence.
The final hybridoma used in this study was cloned two times by limiting dilution. The monoclonal antibody produced from this clone was determined to be of the IgG2a subclass and is specific for Rab26 Figure 1—figure supplement 1.
The antibody is commercially available from Synaptic Systems Göttingen, Germany. Cy3-labeled goat anti-mouse or anti-rabbit and Alexa labeled goat anti-mouse secondary antibodies were purchased from Dianova Hamburg, Germany and used at a dilution of Horseradish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies were obtained from Bio-Rad Hercules, CA and used at a dilution of or Likewise, inserts encoding Rab26 QL, T77N or NI mutants were generated by recombinant PCR and similarly inserted into these vectors.
For recombinant protein expression in bacteria, inserts for the Rab26 variants were inserted into pGEX-KG using EcoRI and BamHI while the insert encoding alpha-GDI was sub-cloned into pETa Novagen, Madison, WI.
The sequence corresponding to murine Atg16L1 1— BC was cloned into pETa Novagen using NdeI and NotI restriction sites. Full-length murine Atg5 1— BC was cloned with an N-terminal thrombin cleavage site into the multiple cloning site 1 of pETDuet-1 Novagen using the SalI and NotI sites. The vector expressing neuropeptide Y NPY was generated by inserting the sequence encoding human pro-NPY into the pmRFP vector.
Cloning was performed according to standard procedures Janssen, The plasmid expressing GFP-tagged human LC3B was a kind gift from Dr Zvulun Elazar Weizmann Institute, Israel.
Culturing of the HEK and HeLa SS6 cell lines and the preparation of high density primary rat hippocampal neurons have been previously described Rosenmund and Stevens, ; Chua et al. Neurons were transfected between 7 to 12 days after seeding or, in the case of the cell lines, 1 day after seeding using Lipofectamine Invitrogen, Carlsbad, CA according to the manufacturer's protocol.
Neurons in Figures 3F, 4 , Figure 3—figure supplement 1 and Figure 4—figure supplement 1 were transfected using calcium phosphate as previously described Pavlos et al.
Immunostaining was then performed as described in Chua et al. Afterwards, cells were permeabilized with 0. Incubation with primary antibodies diluted in blocking solution was then carried out for 1 hr at room temperatures or overnight at 4°C.
Subsequently, cells were exposed to secondary Cy3 or Alexafluor conjugated goat anti-rabbit and anti-mouse antibodies, respectively, for 1 hr at room temperature. After washing, cells were mounted on slides SuperFrost Plus, VWR International bvba, Leuven, Belgium and then imaged using a confocal microscope LSM , Zeiss, Germany or an epifluorescence microscope Axiovert M, Zeiss, Germany.
Linescan analyses were performed using ImageJ or LAS AF Lite software. To visualize synaptic vesicles that have undergone recycling, live neurons transfected with EGFP-Rab26WT were incubated in culture for 24 hr with Oyster labeled anti-synaptotagmin-I antibodies Synaptic Systems that recognize its luminal domain Willig et al.
The UAST-YFP. Rab26, UAST-YFP. Rab26QL, UAST-YFP. Rab26TN Zhang et al. Dissection and immunostaining of neuromuscular junctions from third instar larvae were performed as described Schmid and Sigrist, using the following antibodies: mouse Anti-Brp hybridoma clone nc82, DSHB; dilution , anti-Csp antibody hybridoma clone ab49, DSHB; dilution , the chicken anti-GFP antibody Abcam; dilution and the goat anti-HRP Sigma; dilution.
Dylight labeled anti-goat and Alexa labeled anti-chicken secondary antibodies were purchased from Jackson ImmunoResearch Laboratories West Grove, PA. Alexa conjugated anti-mouse secondary antibodies were purchased from Invitrogen Carlsbad, CA.
Metrics details. Autophagy and GTPases is ad conserved cellular Autohpagy process in eukaryotes GTPPases facilitates the Autophagy and GTPases and reutilization anf damaged organelles and compartments. It Tips for proper rehydration a Immune-boosting herbs role in cellular homeostasis, pathophysiological processes, and diverse diseases in humans. Autophagy involves dynamic crosstalk between different stages associated with intracellular vesicle trafficking. Golgi apparatus is the central organelle involved in intracellular vesicle trafficking where Golgi-associated Rab GTPases function as important mediators. This review focuses on the recent findings that highlight Golgi-associated Rab GTPases as master regulators of autophagic flux.
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